Novel receptor nucleic acids and polypeptides

ABSTRACT

Disclosed are nucleic acids encoding BAFF-R polypeptides, as well as antibodies to BAFF-R polypeptides and pharmaceutical compositions including the same. Methods of treating tumorigenic and autoimmune conditions using the nucleic acids, polypeptides, antibodies and pharmaceutical compositions of this invention are also provided.

This application is a divisional of U.S. application Ser. No. 10/380,703(incorporated herein by reference), filed Jul. 28, 2003, which is aNational Stage Entry of PCT/US01/28006, filed Sep. 6, 2001, which claimsthe benefit of U.S. Provisional Application No. 60/312,185, filed Aug.14, 2001, U.S. Provisional Application No. 60/268,499, filed Feb. 13,2001, U.S. Provisional Application No. 60/234,140, filed Sep. 21, 2000,and U.S. Provisional Application No. 60/233,152, filed Sep. 18, 2000.

FIELD OF THE INVENTION

The present invention provides a novel receptor protein. The inventiongenerally relates to nucleic acids and polypeptides. The inventionrelates more particularly to nucleic acids encoding polypeptides relatedto a receptor to BAFF, a B-cell activating factor belonging to the TumorNecrosis Factor (“TNF”) family, which is associated with the expressionof B-cells and immunoglobulins. This receptor can be employed in thetreatment of cancers, lymphomas, autoimmune diseases or inheritedgenetic disorders involving B-cells.

BACKGROUND OF THE INVENTION

The present invention relates to a novel receptor in the TNF family. Anovel receptor has been identified as the BAFF receptor (“BAFF-R”).

The TNF family consists of pairs of ligands and their specific receptorsreferred to as TNF family ligands and TNF family receptors (Bazzoni andBeutler (1996) N. Engl. J. Med. 334(26):1717-1725. The family isinvolved in the regulation of the immune system and possibly othernon-immunological systems. The regulation is often at a “master switch”level such that TNF family signaling can result in a large number ofsubsequent events best typified by TNF. TNF can initiate the generalprotective inflammatory response of an organism to foreign invasion thatinvolves the altered display of adhesion molecules involved in celltrafficking chemokine production to drive specific cells into specificcompartments, and the priming of various effector cells. As such, theregulation of these pathways has clinical potential.

Induction of various cellular responses mediated by such TNF familycytokines is believed to be initiated by their binding to specific cellreceptors. At least two distinct TNF receptors of approximately 55 kDa(TNFR1) and 75 kDa (TNFR2) have been identified (Hohman et al. (1989) J.Biol. Chem. 264:14927-14934; and Brockhaus et al. (1990) Proc. Natl.Acad. Sci. USA 87:3127-3131). Extensive polymorphisms have beenassociated with both TNF receptor genes. Both TNFRs share the typicalstructure of cell surface receptors including extracellular,transmembrane and intracellular domains. The extracellular portion oftype 1 and type 2 TNFRs contains a repetitive amino acid sequencepattern of four cysteine

rich domains (CDRs). A similar repetitive pattern of CDRs exist inseveral other cell surface proteins, including p75 nerve growth factorreceptor, the B-cell antigen CD40 amongst others.

The receptors are powerful tools to elucidate biological pathwaysbecause of their easy conversion to immunoglobulin fusion proteins.These dimeric soluble receptor forms are good inhibitors of eventsmediated by either secreted or surface bound ligands. By binding tothese ligands they prevent the ligand from interacting with cellassociated receptors that can signal. Not only are these receptor-Fcfusion proteins useful in an experimental sense, but they have beensuccessfully used clinically in the case of TNF-R-Fc to treatinflammatory bowel disease, rheumatoid arthritis and the acute clinicalsyndrome accompanying OKT3 administration (Eason et al. (1996)Transplantation 61(2):224-228; Feldmann et al. (1996) Int. Arch. AllergyImmunol. 111(4):362-365; and van Dullemen et al. (1995) Gastroenterol.109(1):129-135). One can envision that manipulation of the many eventsmediated by signaling through the TNF family of receptors will have wideapplication in the treatment of immune based diseases and also the widerange of human diseases that have pathological sequelae due to immunesystem involvement. A soluble form of a recently described receptor,osteoprotegerin, can block the loss of bone mass and, therefore, theevents controlled by TNF family receptor signaling are not necessarilylimited to immune system regulation (Simonet et al. (1997) Cell89(2):309-319). Antibodies to the receptor can block ligand binding andhence can also have clinical application. Such antibodies are often verylong-lived and may have advantages over soluble receptor-Fc fusionproteins which have shorter blood half-lives.

While inhibition of the receptor mediated pathway represents the mostexploited therapeutic application of these receptors, originally it wasthe activation of the TNF receptors that showed clinical promise(Aggarwal and Natarajan (1996) Eur Cytokine Netw. 7(2):93-124).Activation of the TNF receptors can initiate cell death in the targetcell and hence the application to tumors was and still is attractive(Eggermont et al. (1996) Ann. Surg. 224(6):756-765). The receptor can beactivated either by administration of the ligand, i.e. the naturalpathway or some antibodies that can crosslink the receptor are alsopotent agonists. Antibodies would have an advantage in oncology sincethey can persist in the blood for long periods whereas the ligandsgenerally have short lifespans in the blood. As many of these receptorsmay be expressed more selectively in tumors or they may only signal celldeath or differentiation in tumors, agonist antibodies could be goodweapons in the treatment of cancer. Likewise, many positiveimmunological events are mediated via the TNF family receptors, e.g.host inflammatory reactions, antibody production etc. and thereforeagonistic antibodies could have beneficial effects in other,non-oncological applications.

Paradoxically, the inhibition of a pathway may have clinical benefit inthe treatment of tumors. For example the Fas ligand is expressed by sometumors and this expression can lead to the death of Fas positivelymphocytes thus facilitating the ability of the tumor to evade theimmune system. In this case, inhibition of the Fas system could thenallow the immune system to react to the tumor in other ways now thataccess is possible (Green and Ware (1997) Proc. Natl. Acad. Sci. USA94(12):5986-90).

The TNF family ligand BAFF, also known as TALLY-1, THANK, BLyS and zTNF4(Schneider et al. (1999) J. Exp. Med. 189(11):1747-1756; Shu et al.(1999) J. Leukoc. Biol. 65(5):680-683; Mukhopadhyay et al. (1999) J.Biol. Chem. 274(23):15978-15981; Moore et al. (1999) Science285(5425):260-263; Gross et al (2000) Nature 404(6781):995-999) enhancesB cell survival in vitro (Batten et al. (2000) J. Exp. Med.192(10):1453-1466) and has emerged as a key regulator of peripheral Bcell populations in vivo. Mice over-expressing BAFF display mature Bcell hyperplasia and symptoms of systemic lupus erythaematosus (SLE)(Mackay et al. (1999) J. Exp. Med. 190(11):1697-1710). As well, some SLEpatients have significantly increased levels of BAFF in their serum(Zhang et al. (2001) J. Immunol. 166(1):6-10). It has therefore beenproposed that abnormally high levels of this ligand may contribute tothe pathogenesis of autoimmune diseases by enhancing the survival ofautoreactive B cells (Batten et al. (2000) J. Exp. Med.192(10):1453-1466).

BAFF, a type II membrane protein, is produced by cells of myeloid originSchneider et al. (1999) J. Exp. Med. 189(11): 1747-1756; Moore et al.(1999) Science 285(5425):260-263) and is expressed either on the cellsurface or in a soluble form (Schneider et al. (1999) J. Exp. Med.189(11):1747-1756). Two TNF receptor family members, BCMA and TACI havepreviously been shown to interact with BAFF (Gross et al. (2000) Nature404:995-999; Thompson et al. (2000) J. Exp. Med. 192(1):129-135; Xia etal. (2000) J. Exp. Med. 192:137-143; Marsters et al. (2000) Curr. Biol.10(13):785-788; Shu et al. (2000) J. Leukoc. Biol. 65(5):680-683; Wu etal. (2000) J. Biol. Chem. 275:35478-35485).

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the discovery of “BAFF-R,”a BAFF receptor protein, polynucleotide sequences and the BAFF-Rpolypeptides encoded by these nucleic acid sequences.

In one aspect, the invention provides an isolated nucleic acid whichencodes a BAFF-R polypeptide, or a fragment or derivative thereof. Thenucleic acid can include, e.g. nucleic acid sequence encoding apolypeptide at least 50% identical, or at least 90% identical, to apolypeptide comprising the amino acid sequence of FIG. 2D (SEQ ID NO:5).

The invention also provides a substantially pure nucleic acid moleculecomprising a sequence that hybridizes under stringent conditions to ahybridization probe, the nucleic acid sequence of the probe consistingof the coding sequence of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4)or the complement of said coding sequence.

In some embodiments, the nucleic acid sequence encodes a polypeptidehaving the sequence of FIG. 2D (SEQ ID NO:5) with at least oneconservative amino acid substitution.

In some embodiments, the nucleic acid sequence encodes a polypeptidethat binds BAFF.

The nucleic acid can include, e.g., a nucleic acid which includes thenucleotide sequence shown in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ IDNO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4) and FIG. 3 (SEQ IDNO:6).

The nucleic acid can be, e.g., a genomic DNA fragment, or it can be acDNA molecule. Also included in the invention is a vector containing oneor more of the nucleic acids described herein, and a cell containing thevectors or nucleic acids described herein.

In another aspect, the invention provides a substantially pure nucleicacid molecule encoding a fusion protein comprising at least twosegments, wherein one of the segments comprises a polypeptide orfragment thereof as described in the amino acid sequences set forth inthe above embodiments of the invention. The invention also provides afusion protein comprising at least two or three segments, wherein thefirst segment comprises a heterologous signal polypeptide, the secondcomprises a polypeptide or fragment thereof as described in the BAFF-Ramino acid sequences set forth in the above embodiments of the inventionand the third segment comprises an immunoglobulin polypeptide.Alternatively, the first segment comprises an immunoglobulin polypeptidefragment containing a signal sequence and the second segment comprisesthe BAFF-R polypeptide fragment.

In other aspects, the invention provides a substantially pure bindingagent that specifically binds to the polypeptide of the above-statedembodiments of the invention.

The present invention is also directed to host cells transformed with arecombinant expression vector comprising any of the nucleic acidmolecules described above.

In another aspect, the invention includes a pharmaceutical compositionthat includes a BAFF-R nucleic acid and a pharmaceutically acceptablecarrier or diluent.

In a further aspect, the invention includes a substantially purifiedBAFF-R polypeptide, e.g., any of the polypeptides encoded by a BAFF-Rnucleic acid.

The invention also includes a pharmaceutical composition that includes aBAFF-R polypeptide and a pharmaceutically acceptable carrier or diluent.

In a still further aspect, the invention provides an antibody that bindsspecifically to a BAFF-R polypeptide. The antibody can be, e.g., amonoclonal or polyclonal antibody. The invention also includes apharmaceutical composition including BAFF-R antibody and apharmaceutically acceptable carrier or diluent. The present invention isalso directed to isolated antibodies that bind to an epitope on apolypeptide encoded by any of the nucleic acid molecules describedabove.

The present invention is further directed to kits comprising antibodiesthat bind to a polypeptide encoded by any of the nucleic acid moleculesdescribed above and a negative control antibody.

The invention further provides a method for producing a BAFF-Rpolypeptide. The method includes providing a cell containing a BAFF-Rnucleic acid, e.g., a vector that includes a BAFF-R nucleic acid, andculturing the cell under conditions sufficient to express the BAFF-Rpolypeptide encoded by the nucleic acid. The expressed BAFF-Rpolypeptide is then recovered from the cell. Preferably, the cellproduces little or no endogenous BAFF-R polypeptide. The cell can be,e.g., a prokaryotic cell or eukaryotic cell.

The present invention provides a method of inducing an immune responsein a mammal against a polypeptide encoded by any of the nucleic acidmolecules disclosed above by administering to the mammal an amount ofthe polypeptide sufficient to induce the immune response.

The present invention is also directed to methods of identifying acompound that binds to BAFF-R polypeptide by contacting the BAFF-Rpolypeptide with a compound and determining whether the compound bindsto the BAFF-R polypeptide.

The present invention is also directed to methods of identifying acompound that binds a nucleic acid molecule encoding BAFF-R polypeptideby contacting BAFF-R nucleic acid with a compound and determiningwhether the compound binds the nucleic acid molecule.

The invention further provides methods of identifying a compound thatmodulates the activity of a BAFF-R polypeptide by contacting BAFF-Rpolypeptide with a compound and determining whether the BAFF-Rpolypeptide activity is modified.

The present invention is also directed to compounds that modulate BAFF-Rpolypeptide activity identified by contacting a BAFF-R polypeptide withthe compound and determining whether the compound modifies activity ofthe BAFF-R polypeptide, binds to the BAFF-R polypeptide, or binds to anucleic acid molecule encoding a BAFF-R polypeptide.

In another aspect, the invention provides a method of diagnosing aB-cell mediated condition, e.g., an autoimmune disorder or cancer, in asubject. The method includes providing a protein sample from the subjectand measuring the amount of BAFF-R polypeptide in the subject sample.The amount of BAFF-R in the subject sample is then compared to theamount of BAFF-R polypeptide in a control protein sample. An alterationin the amount of BAFF-R polypeptide in the subject protein samplerelative to the amount of BAFF-R polypeptide in the control proteinsample indicates the subject has a BAFF-R mediated condition. A controlsample is preferably taken from a matched individual, i.e., anindividual of similar age, sex, or other general condition but who isnot suspected of having the condition. Alternatively, the control samplemay be taken from the subject at a time when the subject is notsuspected of having the disorder. In some embodiments, the BAFF-Rpolypeptide is detected using a BAFF-R antibody.

In a further aspect, the invention includes a method of diagnosing aB-cell mediated condition, e.g., autoimmune disorder in a subject. Themethod includes providing a nucleic acid sample, e.g., RNA or DNA, orboth, from the subject and measuring the amount of the BAFF-R nucleicacid in the subject nucleic acid sample. The amount of BAFF-R nucleicacid sample in the subject nucleic acid is, then compared to the amountof BAFF-R nucleic acid in a control sample. An alteration in the amountof BAFF-R nucleic acid in the sample relative to the amount of BAFF-R inthe control sample indicates the subject has an autoimmune condition.

In a further aspect, the invention includes a method of diagnosing atumorigenic or autoimmune condition in a subject. The method includesproviding a nucleic acid sample from the subject and identifying atleast a portion of the nucleotide sequence of a BAFF-R nucleic acid inthe subject nucleic acid sample. The BAFF-R nucleotide sequence of thesubject sample is then compared to a BAFF-R nucleotide sequence of acontrol sample. An alteration in the BAFF-R nucleotide sequence in thesample relative to the BAFF-R nucleotide sequence in said control sampleindicates the subject has such a condition.

In a still further aspect, the invention provides method of treating orpreventing or delaying a B-cell mediated condition. The method includesadministering to a subject in which such treatment or prevention ordelay is desired a BAFF-R nucleic acid, a BAFF-R polypeptide, or ananti-BAFF-R antibody in an amount sufficient to treat, prevent, or delaya tumorigenic or immunoregulatory condition in the subject.

The conditions diagnosed, treated, prevented or delayed using the BAFF-Rnucleic acid molecules, polypeptides or antibodies can be a cancer or animmunoregulatory disorder. Diseases include those that are autoimmune innature such as systemic lupus erythematosus, rheumatoid arthritis,myasthenia gravis, autoimmune hemolytic anemia, idiopathicthrombocytopenia purpura, anti-phospholipid syndrome, Chagas' disease,Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa andrapidly progressive glomerulonephritis. The therapeutic agent also hasapplication in plasma cell disorders such as multiple myeloma,Waldenstrom's macroglobulinemia, heavy-chain disease, primary orimmunocyte-associated amyloidosis, and monoclonal gammopathy ofundetermined significance (MGUS). Oncology targets include B cellcarcinomas, leukemias, and lymphomas.

Compositions and methods of treatment using the nucleic acids,polypeptides and antibodies of the present invention can be used withany condition associated with undesired cell proliferation. Inparticular, the present invention can be used to treat tumor cells whichexpress BAFF and/or BAFF-R.

Compositions of the invention comprising BAFF-R agonists (such asantibodies that bind to BAFF-R and mimic BAFF) also may be used to treatimmune deficiencies marked by low amounts of B cells, for example. Suchdisorders may be caused by radiation and/or chemotherapy, for example.

In another aspect of the invention a method for decreasing aggregationof a recombinantly expressed protein is provided. The method comprisescomparison of homologs of a protein or fusion protein thereof todetermine conserved domains and non-identical amino acids withinconserved regions. Generally, at least one non-polar amino acid ischanged to an uncharged polar amino acid or to a proline, alanine orserine.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the DNA sequence of the BJAB cDNA (SEQ ID NO:1) cloned inpJST576.

FIG. 1B shows the complete DNA sequence of the cDNA of the IMAGE clone2000271 (EST AI250289) (SEQ ID NO:2).

FIG. 2A shows the nucleotide sequence of JST576 with an intron removedas predicted by the GENESCAN program (SEQ ID NO:3).

FIG. 2B shows a 1% agarose gel of PCR products obtained for BAFF-R usingeither first strand cDNA generated from BJAB or IM-9 RNA or on JST576cDNA. Lane 1. Lambda DNA HindIII digest. Lane 2. BJAB oligo dT primedBAF-225/BAF-191. Lane 3. BJAB oligo dT primed BAF-226/BAF-191. Lane 4.BJAB random primed BAF-225/BAF-191. Lane 5. BJAB random primedBAF-226/BAF-191. Lane 6. IM-9 oligo dT primed BAF-225/BAF-191. Lane 7.IM-9 oligo dT primed BAF-226/BAF-191. Lane 8. IM-9 random primedBAF-225/BAF-191. Lane 9. IM-9 random primed BAF-2-26/BAF-191. Lane 10.JST576 cDNA BAF-225/BAF-191. Lane 11. JST576 cDNA BAF-226/BAF-191. Lane12. No template BAF-225/BAF-191. Lane 13. No template BAF-226/BAF-191.

FIG. 2C shows the mature JST576 (BAFF-R) sequence (SEQ ID NO:4) (alsoGenBank Accession No. AF373846) determined by sequencing bulk PCRproduct flag the predicted intron from BJAB first strand cDNA.

FIG. 2D shows the amino acid sequence of BAFF-R (JST576) (SEQ ID NO:5).The A (Ala) residue in bold indicates the sequence resulting from theuse of the alternative splice acceptor site. The predicted transmembranedomain is boxed and the putative stop transfer signal is underlined.

FIG. 3 depicts the spliced version of JST576 (SEQ ID NO:6) containing 5′UTR sequence obtained by RT-PCR from human spleen first strand cDNA, andthe deduced amino acid sequence (SEQ ID NO:7). This sequence contains anupstream stop codon in frame with the ATG.

FIG. 4A shows the sequence of the murine BAFF-R cDNA (SEQ BD NO:8) (alsoGenBank Accession No. AF373847).

FIG. 4B shows the amino acid sequence of murine BAFF-R (SEQ ID NO:9).The Cys residues are bold and underlined and the predicted transmembraneregion is boxed.

FIG. 4C shows the homology between human (SEQ ID NO:10) and murine (SEQID NO:9) BAFF-R protein sequences.

FIG. 5 depicts human BAFF binding to JST576 transfected cells. 293EBNAcells were co-transfected with pJST576 or CA336 (huTACI) and a GFPreporter construct. Cells assayed for BAFF binding with 1 ug/mlbiotinylated myc-huBAFF followed by SAV-PE.

FIG. 6 shows human and murine BAFF binding to JST576 transfected cells.293EBNA cells were co-transfected with pJST576 and a GFP reporterconstruct. Cells assayed 24 hr later for BAFF binding with 5 ug/mlflag-huBAFF or flag-muBAFF followed anti-flag monoclonal antibody M2 anddonkey anti-mouse IgG-PE.

FIG. 7 shows that APRIL does not bind to JST576 transfected cells.293EBNA cells were co-transfected with pJST576 or CA336 (huTACI) and aGFP reporter construct. Cells were assayed for APRIL binding with 1ug/ml myc-muAPRIL followed by anti-muAPRIL rat-IgG2b, biotinylatedanti-rat FcG2b, and SAV-PE.

FIG. 8 shows that BAFF precipitates a protein from JST576transfected-cells. 293EBNA cells were transfected with either BAFF-R(JST576), vector only CH269) or huTACI (CA336) and pulsed with ³⁵Scysteine and methionine. Extracts were immunoprecipitated withflag-huBAFF and run on a reducing SDS-PAGE-gel. Molecular weight markersare indicated at left.

FIG. 9 depicts the nucleic acid sequence (SEQ ID NO:11) and its derivedamino acid sequence (SEQ ID NO:12) of a gene encoding a human BAFF-R:Fc:nucleic acid residues 1-63 encode the murine IgG-kappa signal sequence;nucleic acid residues 64-66 were used to introduce a restriction enzymesite, nucleic acid residues 67-276 encode part of the BAFF-Rextracellular domain, nucleic acid residues 277-279 were used tointroduce a restriction enzyme site, and nucleic acid residues 280-960encode the Fc region of human IgG1.

FIG. 10 depicts the results of Northern blot analysis using the EcoNIfragment of JST56 as a probe. All exposures are 4 days. 10A: Clontechhuman Immune II blot; 10B: Clontech human 12 lane multi-tissue blot;10C: Clontech human multi-tissue B blot.

FIG. 11 shows the result of Northern blot analysis of 20 μg of total RNAisolated from various cell lines. The blot was probed with an EcoNIrestriction fragment of JST576 and exposed for 4 days. The ability ofthe cell lines to bind BAFF, as determined by FACS analysis, isindicated below the lane.

FIG. 12 shows the results of immunoprecipitation results usingBAFF-R:Fc. Human BAFF is immunoprecipitated with BAFF-R:Fc or BCMA:Fc,but not Fn14-Fc. Control BAFF protein is shown in lane 1.

FIG. 13 shows that human BAFF-R:Fc blocks human BAFF binding to BJABcells. The results of FACS analysis are shown in FIG. 13A. Curve Erepresents biotinylated BAFF binding to BJAB cells in the absence ofBAFF-R:Fc. Curves B-D represent the ability of BAFF to bind to BJABcells in the presence of 5 ug/ml, 1 ug/ml or 0.2 ug/ml, respectively.Curve A is the second step only curve. FIG. 13B illustrates the abilityof various concentrations of BAFF-R:Fc (squares) compared to TACI:Fc(triangles) or a nonspecific fusion protein, LT_R:Fc (circles), to blockthe binding of BAFF to the receptor expressing BJAB cells.

FIG. 14 shows the ability of BAFF-R:Fc to block BAFF-inducedco-stimulation of splenic B cells. A graph of [³H] thymidineincorporation (cpm) versus increasing amounts of hBAFF (ng/ml) is shown.

FIG. 15 shows that BAFF-R:Fc1 treatment results in a loss of peripheralB cells in normal mice.

FIG. 16 shows that treatment of mice with human and mouseBAFF-R:Fc-reduces the number of splenic B220+ B cells.

FIG. 17 shows that administration of BAFF-R:Fc to mice reduces thepercentage of lymph node B220+ B cells.

FIG. 18 shows that administration of BAFF-R:Fc to mice reducesperipheral blood B220+ B cells.

FIG. 19A shows FACS data from supernatants of four clones that produceantibodies that bind BAFF-R. Also shown is control supernatant whichdoes not contain antibodies that binds BAFF-R.

FIG. 19B shows a histogram showing that two clones that block BAFFbinding to BAFF-R. (a) shows the no BAFF control; (b) shows the blockingability of the antibody from clone 2; (c) shows the blocking ability ofthe antibody from clone 9; and (d) shows the curve from a controlantibody that does not bind BAFF-R.

FIG. 20 shows an alignment of the amino acid sequences of humanBAFF-R:Fc (hBAFF-R) and mouse BAFF-R:Fc (mBAFF-R) extracellular domainsand the percentage of aggregation observed upon expression of the Fcfusion proteins containing the indicated sequences. Numbered JST clonesrepresent the amino acid sequences showing mutations (shown inunderline) in the parent sequences and the resulting aggregation ofexpressed protein. Shown are the partial sequences for human (aminoacids 2-71 of SEQ ID NO:1O; SEQ ID NO:13) and mouse (amino acids 2-71 ofSEQ ID NO:9; SEQ ID NO:14) BAFF-R; and the corresponding portions forthe following clones: JST659 (SEQ ID NO:15), JST66O (SEQ ID NO:16,JST661 (SEQ ID NO:17), JST662 (SEQ ID NO:18), JST663 (SEQ ID NO:19),JST673 (SEQ ID NO:20), JST674 (SEQ ID NO:21), JST675 (SEQ ID NO:22),JST672 (SEQ ID NO:23), JST676 (SEQ ID NO: 24), JST671 (SEQ ID NO:25),JST677 (SEQ ID NO:26), JST678 (SEQ ID NO:27), JST664 (SEQ ID NO:28),JST668 (SEQ ID NO:29), JST665 (SEQ ID NO:30), JST666 (SEQ ID NO: 31),and JST667 (SEQ ID NO:32).

FIG. 21 shows an autoradiograph of proteins immunoprecipitated usinglysates prepared from BAFF-R-i.c.d. (BAFF-R intracellular domain) (lane1), or control vector-(lane 2) transfected cells. Approximately 6×10⁶293E cells were transfected with a construct encoding BAFFR-i.c.d. ormock plasmid. After 48 hours, the cells were metabolically labeled with³⁵S for 24 hours, lysed with lysis buffer, precleared, andimmunoprecipitated with an antimyc mAb, 9E10. The immunoprecipitateswere separated by 10-20% SDS PAGE under reducing condition.

DETAILED DESCRIPTION OF THE INVENTION

The reference works, patents, patent applications, and scientificliterature, including accession numbers to GenBank database sequences,that are referred to herein establish the knowledge of those with skillin the art and are hereby incorporated by reference in their entirety tothe same extent as if each was specifically and individually indicatedto be incorporated by reference. Any conflict between any referencecited herein and the specific teachings of this specification shall beresolved in favor of the latter. Likewise, any conflict between anart-understood definition of a word or phrase and a definition of theword or phrase as specifically taught in this specification shall beresolved in favor of the latter.

Standard reference works setting forth the general principles ofrecombinant DNA technology known to those of skill in the art includeAusubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York (1998); Sambrook et al. MOLECULAR CLONING: A LABORATORYMANUAL, 2D ED., Cold Spring Harbor Laboratory Press, Plainview, N.Y.(1989); Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODSIN BIOLOGY AND MEDICINE, CRC Press, Boca Raton (1995); McPherson, Ed.,DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford (1991).

The present invention discloses BAFF-R nucleic acids, isolated nucleicacids that encode BAFF-R polypeptide or a portion thereof, BAFF-Rpolypeptides, vectors containing these nucleic acids, host cellstransformed with the BAFF-R nucleic acids, anti-BAFF-R antibodies, andpharmaceutical compositions. Also disclosed are methods of making BAFF-Rpolypeptides, as well as methods of screening, diagnosing, treatingconditions using these compounds, and methods of screening compoundsthat modulate BAFF-R polypeptide activity.

The BAFF-R nucleic acids and polypeptides, as well as BAFF-R antibodies,as well as pharmaceutical compositions discussed herein, are useful,inter alia, in treating cancer and/or immunoregulatory conditions. Thesedisorders include, e.g., B cell-mediated diseases that are autoimmune innature such as systemic lupus erythematosus, rheumatoid arthritismyasthenia gravis, autoimmune hemolytic anemia, idiopathicthrombocytopenia purpura, anti-phospholipid syndrome, Chagas' disease,Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa andrapidly progressive glomerulonephritis. This therapeutic agent also hasapplication in plasma cell disorders such as multiple myeloma,Waldenstrom's macroglobulinemia, heavy-chain disease, primary orimmunocyte-associated amyloidosis, and monoclonal gammopathy ofundetermined significance (MGUS). Oncology targets include B cellcarcinomas, leukemias, and lymphomas.

BAFF-R Nucleic Acids

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode BAFF-R proteins or biologically active portions thereof.Also included are nucleic acid fragments sufficient for use ashybridization probes to identify BAFF-R-encoding nucleic acids (e.g.,BAFF-R mRNA) and fragments for use as polymerase chain reaction (PCR)primers for the amplification or mutation of BAFF-R nucleic acidmolecules. As used herein, the term “nucleic acid molecule” is intendedto include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules(e.g., mRNA), analogs of the DNA or RNA generated using nucleotideanalogs, and derivatives, fragments and homologs thereof. The nucleicacid molecule can be single-stranded or double-stranded, but preferablyis double-stranded DNA.

“Probes” refer to nucleic acid sequences of variable length, preferablybetween at least about 10 nucleotides (nt) or as many as about, e.g.,6,000 nt, depending on use. Probes are used in the detection ofidentical, similar, or complementary nucleic acid sequences. Longerlength probes are usually obtained from a natural or recombinant source,are highly specific and much slower to hybridize than oligomers. Probesmay be single- or double-stranded and designed to have specificity inPCR, membrane-based hybridization technologies, or ELISA-liketechnologies.

An “isolated” nucleic acid molecule is one that is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Examples of isolated nucleic acid molecules include, butare not limited to, recombinant DNA molecules contained in a vector,recombinant DNA molecules maintained in a heterologous host cell,partially or substantially purified nucleic acid molecules, andsynthetic DNA or RNA molecules. Preferably, an “isolated” nucleic acidis free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated BAFF-R nucleic acidmolecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial or culture medium when produced by recombinant techniques, orof chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of FIG. 1A (SEQ ID NO:1), FIG.1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4) and FIG.3 (SEQ ID NO:6), or a complement of any of these nucleotide sequences,can be isolated using standard molecular biology techniques and thesequence information provided herein. Using all or a portion of thenucleic acid sequences of FIGS. 1A, B, 2A, C and 3 as a hybridizationprobe, BAFF-R nucleic acid sequences can be isolated using standardhybridization and cloning techniques (e.g., as described in Sambrook etal., Eds., MOLECULAR CLONING: A LABORATORY MANUAL 2ND ED., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, etal., Eds., CURRET PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NewYork, N.Y., 1993).

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to BAFF-R nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

As used herein, the term “oligonucleotide” refers to a series of linkednucleotide residues, which oligonucleotide has a sufficient number ofnucleotide bases to be used in a PCR reaction. A short oligonucleotidesequence may be based on, or designed from, a genomic or cDNA sequenceand is used to amplify, confirm, or reveal the presence of an identical,similar or complementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise portions of a nucleic acid sequence having atleast about 10 nt and as many as 50 nt, preferably about 15 nt to 30 nt.They may be chemically synthesised and may be used as probes.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule that is a complement of thenucleotide sequence shown in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ IDNO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ IDNO:6). In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule that is a complement of thenucleotide sequence shown in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ IDNO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ IDNO:6), or a portion of this nucleotide sequence. A nucleic acid moleculethat is complementary to the nucleotide sequence shown in FIG. 1A (SEQID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C(SEQ IDNO:4), and FIG. 3 (SEQ ID NO:6) is one that is sufficientlycomplementary to the nucleotide sequence shown in FIG. 1A (SEQ ID NO:1),FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C(SEQ ID NO:4), andFIG. 3 (SEQ ID NO:6) that it can hydrogen bond with little or nomismatches to the nucleotide sequence shown in FIG. 1A (SEQ ID NO:1,FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4) andFIG. 3 (SEQ ID NO:6) thereby forming a stable duplex.

As used herein, the term “complementary” refers to Watson-Crick orHoogsteen base paining between nucleotides units of a nucleic acidmolecule, and the term “binding” means the physical or chemicalinteraction between two polypeptides or compounds or associatedpolypeptides or compounds or combinations thereof. Binding includesionic, non-ionic, Van der Waals, hydrophobic interactions, etc. Aphysical interaction can be either direct or indirect. Indirectinteractions may be through or due to the effects of another polypeptideor compound. Direct binding refers to interactions that do not takeplace through, or due to, the effect of another polypeptide or compound,but instead are without other substantial chemical intermediates.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of FIG. 1A (SEQ ID NO:1), FIG. 1B(SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3(SEQ ID NO:6), e.g. a fragment that can be used as a probe or primer ora fragment encoding a biologically active portion of BAFF-R. Fragmentsprovided herein are defined as sequences of at least 6 (contiguous)nucleic acids or at least 4 (contiguous) amino acids, a lengthsufficient to allow for specific hybridization in the case of nucleicacids or for specific recognition of an epitope in the case of aminoacids, respectively, and are at most some portion less than a fulllength sequence. Fragments may be derived from any contiguous portion ofa nucleic acid or amino acid sequence of choice. Derivatives are nucleicacid sequences or amino acid sequences formed from the native compoundseither directly or by modification or partial substitution. Analogs arenucleic acid sequences or amino acid sequences that have a structuresimilar to, but not identical to, the native compound but differs fromit in respect to certain components or side chains. Analogs may besynthetic or from a different evolutionary origin and may have a similaror opposite metabolic activity compared to wild type.

Derivatives and analogs may be full length or other than fail length, ifthe derivative or analog contains a modified nucleic acid or amino acid,as described below. Derivatives or analogs of the nucleic acids orproteins of the invention include, but are not limited to, moleculescomprising regions that are substantially homologous to the nucleicacids or proteins of the invention, in various embodiments, by at leastabout 45%, 50%, 70%, 80%, 95%, 98%, or even 99% identity (with apreferred identity of 80-99%) over a nucleic acid or amino acid sequenceof identical size or when compared to an aligned sequence in which thealignment is done by a computer homology program known in the art, orwhose encoding nucleic acid is capable of hybridizing to the complementof a sequence encoding the aforementioned proteins under stringent,moderately stringent, or low stringent conditions. See e.g. Ausubel, etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NewYork, N.Y., 1993, and below. An exemplary program is the Gap program(Wisconsin Sequence Analysis Package, Version 8 for UNIX, GeneticsComputer Group, University Research Park, Madison, Wis.) using thedefault settings, which uses the algorithm of Smith and Waterman (1981)Adv. Appl. Math. 2:482-489, which is incorporated herein by reference inits entirety).

A “homologous nucleic acid sequence” or “homologous amino acidsequence,” or variations thereof, refer to sequences characterized by ahomology at the nucleotide level or amino acid level as discussed above.Homologous nucleotide sequences encode those sequences coding forisoforms of BAFF-R polypeptide. Isoforms can be expressed in differenttissues of the same organism as a result of, for example, alternativesplicing of RNA. Alternatively, isoforms can be encoded by differentgenes. In the present invention, homologous nucleotide sequences includenucleotide sequences encoding for a BAFF-R polypeptide of species otherthan humans, including, but not limited to, mammals, and thus caninclude, e.g., mouse, rat, rabbit, dog, cat cow, horse, and otherorganisms. Homologous nucleotide sequences also include, but are notlimited to, naturally occurring allelic variations and mutations of thenucleotide sequences set forth herein. A homologous nucleotide sequencedoes not, however, include the nucleotide sequence encoding human BAFF-Rprotein. Homologous nucleic acid sequences include those nucleic acidsequences that encode conservative amino acid substitutions (see below)in FIG. 2D (SEQ ID NO:5) as well as a polypeptide having BAFF-Ractivity. A homologous amino acid sequence does not encode the aminoacid sequence of a human BAFF-R polypeptide.

The nucleotide sequence determined from the cloning of the human BAR-Rgene allows for the generation of probes and primers designed for use inidentifying and/or cloning BAFF-R homologues in other cell types, e.g.,from other tissues, as well as BAFF-R homologues from other mammals. Theprobe/primer typically comprises a substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutivesense strand nucleotide sequence of any of FIG. 1A (SEQ ID NO:1), FIG.1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4) and FIG.3 (SEQ ID NO:6) or an anti-sense strand nucleotide sequence of any ofFIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3),FIGS. 2C (SEQ ID NO:4), and 3 (SEQ ID NO:6) or of a naturally occurringmutant of any of FIG. 1A (SEQ ID NO:1), Fig. B (SEQ ID NO:2), FIG. 2A(SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6).

Probes based on the human BAFF-R nucleotide sequence can be used todetect transcripts or genomic sequences encoding the same or homologousproteins. In various embodiments, the probe further comprises a labelgroup attached thereto, e.g. the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a BAFF-R protein, such as by measuring a levelof a BAFF-R-encoding nucleic acid in a sample of cells from a subjecte.g., detecting BAFF-R mRNA levels or determining whether a genomicBAFF-R gene has been mutated or deleted.

“A polypeptide having a biologically active portion of BAFF-R” refers topolypeptides exhibiting activity similar, but not necessarily identicalto, an activity of a polypeptide of the present invention, includingmature forms, as measured in a particular biological assay, with orwithout dose dependency. A nucleic acid fragment encoding a“biologically active portion of BAFF-R” can be prepared by isolating aportion of any of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A(SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6) thatencodes a polypeptide having a BAFF-R biological activity (biologicalactivities of the BAFF-R proteins are described below), expressing theencoded portion of BAFF-R protein (e.g., by recombinant expression invitro) and assessing the activity of the encoded portion of BAFF-R. Forexample, a nucleic acid fragment encoding a biologically active portionof BAFF-R can optionally include a BAFF binding domain. In anotherembodiment, a nucleic acid fragment encoding a biologically activeportion of BAFF-R includes one or more regions.

BAFF-R Variants

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequences shown in FIG. 1A (SEQ ID NO:1), FIG. 1B(SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIGS. 2C (SEQ ID NO:4), and 3 (SEQID NO:6) due to degeneracy of the genetic code. These nucleic acids thusencode the same BAFF-R protein as that encoded by the nucleotidesequence shown in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A(SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6). Inanother embodiment, an isolated nucleic acid molecule of the inventionhas a nucleotide sequence encoding a protein having an amino acidsequence shown in FIG. 2D (SEQ ID NO:5).

In addition to the human BAFF-R nucleotide sequence shown in any of FIG.1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C(SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6), it will be appreciated by thoseskilled in the art that DNA sequence polymorphisms that lead to changesin the amino acid sequences of BAFF-R may exist within a population(e.g., the human population). Such genetic polymorphism in the BAFF-Rgene may exist among individuals within a population due to naturalallelic variation. As used herein, the terms “gene” and “recombinantgene” refer to nucleic acid molecules comprising an open reading frameencoding a BAFF-R protein, preferably a mammalian BAFF-R protein. Suchnatural allelic variations can typically result in 1-5% variance in thenucleotide sequence of the BAFF-R gene. Any and all such nucleotidevariations and resulting amino acid polymorphisms in BAFF-R that are theresult of natural allelic variation and that do not alter the functionalactivity of BAFF-R are intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding BAFF-R proteins from otherspecies, and thus that have a nucleotide sequence that differs from thehuman sequences of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A(SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6) areintended to be within the scope of the invention. Nucleic acid moleculescorresponding to natural allelic variants and homologues of the BAFF-RcDNAs of the invention can be isolated based on their homology to thehuman BAFF-R nucleic acids disclosed herein using the human cDNAs, or aportion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions. Forexample, a soluble human BAFF-R cDNA can be isolated based on itshomology to human membrane-bound BAFF-R. Likewise, a membrane-boundhuman BAFF-R cDNA can be isolated based on its homology to soluble humanBAFF-R.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of any of FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ IDNO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ IDNO:6). In another embodiment, the nucleic acid is at least 10, 25, 50,100, 250 or 500 nucleotides in length. In another embodiment, anisolated nucleic acid molecule of the invention hybridizes to the codingregion. As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% homologous to each othertypically remain hybridized to each other.

Homologs (i.e., nucleic acids encoding BAFF-R proteins derived fromspecies other than human) or other related sequences (e.g., paralogs)can be obtained by low, moderate or high stringency hybridization withall or a portion of the particular human sequence as a probe usingmethods well known in the art for nucleic acid hybridization andcloning.

As used herein, the phrase “stringent hybridization conditions” refersto conditions under which a probe, primer or oligonucleotide willhybridize to its target sequence, but to no other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures than shorter sequences. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present at excess, at Tm, 50% of theprobes are occupied at equilibrium. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes,primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about60° C. for longer probes, primers and oligonucleotides. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide.

Stringent conditions are known to those skilled in the art and can befound in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequencesat least about, 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous toeach other typically remain hybridized to each other. A non-limitingexample of stringent hybridization conditions is hybridization in a highsalt buffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02%PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNAat 65° C. This hybridization is followed by one or more washes in0.2×SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of theinvention that hybridizes under stringent conditions to the sequence ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6corresponds to a naturally occurring nucleic acid molecule. As usedherein, a “naturally-occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein).

In a second embodiment, a nucleic acid sequence that is hybridizable tothe nucleic acid molecule comprising the nucleotide sequence of any ofFIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3),FIGS. 2C (SEQ ID NO:4), and 3 (SEQ ID NO:6) or fragments, analogs orderivatives thereof, under conditions of moderate stringency isprovided. A non-limiting example of moderate stringency hybridizationconditions are hybridization in 6×SSC, 5× Denhardt's solution, 0.5% SDSand 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one ormore washes in 1×SSC, 0.1% SDS at 37° C. Other conditions of moderatestringency that may be used are well known in the art. See, e.g.,Ausubel et al., Eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, NY, 1993; and Kriegler, GENE TRANSFER AND EXPRESSION, ALABORATORY MANUAL, Stockton Press, NY, 1990.

In a third embodiment, a nucleic acid that is hybridizable to thenucleic acid molecule comprising the nucleotide sequence of any of FIG.1A (SEQ ID NO:1), FIG. 2B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2C(SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6) or fragments, analogs orderivatives thereof, under conditions of low stringency, is provided. Anon-limiting example of low stringency hybridization conditions arehybridization in 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmonsperm DNA, 10% (wt/vol) dextran sulfate at 40° C., followed by one ormore washes in 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDSat 50° C. Other conditions of low stringency that may be used are wellknown in the art (e.g., as employed for cross-species hybridizations).See, e.g., Ausubel et al., Eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, NY, 1993; and Kriegler, GENE TRANSFER AND EXPRESSION,A LABORATORY MANUAL, Stockton Press, NY, 1990; Shilo and Weinberg (1981)Proc. Natl. Acad. Sci. USA 78:6789-6792.

Conservative Mutations

In addition to naturally-occurring allelic variants of the BAFF-Rsequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequence of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4),FIG. 3 (SEQ ID NO:6) thereby leading to changes in the amino acidsequence of the encoded BAFF-R protein, without altering the functionalability of the BAFF-R protein. For example, nucleotide substitutionsleading to amino acid substitutions at “non-essential” amino acidresidues can be made in the sequence of any of FIG. 2A (SEQ ID NO:3),FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6). A “non-essential” aminoacid residue is a residue that can be altered from the wild-typesequence of BAFF-R without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. Forexample, amino acid residues that are conserved among the BAFF-Rproteins of the present invention, are predicted to be particularlyunamenable to alteration.

In addition, amino acid residues that are conserved among family membersof the BAFF-R proteins of the present invention, are also predicted tobe particularly unamenable to alteration. For example, BAFF-R proteinsof the present invention can contain at least one domain that is atypically conserved region in TNF family members. As such, theseconserved domains are not likely to be amenable to mutation. Other aminoacid residues, however, (e.g., those that are not conserved or onlysemi-conserved among members of the BAFF-R proteins) may not beessential for activity and thus are likely to be amenable to alteration.

Another aspect of the invention pertains to nucleic acid moleculesencoding BAFF-R proteins that contain changes in amino acid residuesthat are not essential for activity. Such BAFF-R proteins differ inamino acid sequence from FIG. 2D (SEQ ID NO:5), yet retain biologicalactivity. In one embodiment, the isolated nucleic acid molecularcomprises a nucleotide sequence encoding a protein, wherein the proteincomprises an amino acid sequence at least about 45% homologous to theamino acid sequence of FIG. 2D (SEQ ID NO:5). Preferably, the proteinencoded by the nucleic acid molecule is at least about 60% homologous toFIG. 2D (SEQ ID NO:5), more preferably at least about 70%, 80%, 90%,95%, 98%, and most preferably at least about 99% homologous to FIG. 2D(SEQ ID NO:5).

An isolated nucleic acid molecule encoding a BAFF-R protein homologousto the protein of FIG. 2D can be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3(SEQ ID NO:6) such that one or more amino acid substitutions, additionsor deletions are introduced into the encoded protein.

Mutations can be introduced into FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ IDNO:4), or FIG. 3 (SEQ ID NO:6), for example, by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore predicted non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in BAFF-R is replaced with another amino acid residuefrom the same side chain family. Alternatively, in another embodiment,mutations can be introduced randomly along all or part of a BAFF-Rcoding sequence, such as by saturation mutagenesis, and the resultantmutants can be screened for BAFF-R biological activity to identifymutants that retain activity. Following mutagenesis of any of FIG. 2A(SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), and FIG. 3 (SEQ ID NO:6), theencoded protein can be expressed by any recombinant technology known inthe art and the activity of the protein can be determined.

In one embodiment, a mutant BAFF-R protein can be assayed for (1) theability to form protein:protein interactions with other BAFF-R proteins,other cell-surface proteins, or biologically active portions thereof,(2) complex formation between a mutant BAFF-R protein and a BAFF-Rligand; (3) the ability of a mutant BAFF-R protein to bind to anintracellular target protein or biologically active portion thereof;(e.g., avidin proteins); (4) the ability to bind BAFF; or (5) theability to specifically bind a BAFF-R protein antibody.

The invention provides specific mutants encoding a BAFF-R:Fc polypeptidedesigned to alleviate aggregation of expressed protein while maintainingBAFF binding activity. Such mutants, include, for example, clonesencoding the amino acid sequences of JST661 (SEQ ID NO:17), JST662 (SEQID NO:18), JST663 (SEQ ID NO:19), JST673 (SEQ ID NO:20), JST674 (SEQ IDNO:21), JST675 (SEQ ID NO:22), JST672 (SEQ ID NO:23), JST676 (SEQ IDNO:24), JST671 (SEQ ID NO:25), JST677 (SEQ ID NO:26), and JST678 (SEQ IDNO:27). Other embodiments include mutants encoding a BAFF-R or BAFF-R:Fcpolypeptide that has similar aggregation characteristics to native humanBAFF-R or BAFF-R:Fc polypeptide, but also bind BAFF, including, forexample, sequences comprising the amino acid sequences of JST659 (SEQ IDNO:15), JST660 (SEQ ID NO:16), JST664 (SEQ ID NO:28), JST668 (SEQ IDNO:29), JST665 (SEQ ID NO:30), JST666 (SEQ ID NO:31), and JST667 (SEQ IDNO:32). Other embodiments include mutants encoding a BAFF-R or BAFF-R:Fcpolypeptide wherein conserved amino acids between human and mouse BAFF-Rare changed to other conserved amino acids and wherein the bindingactivity of BAFF-R or BAFF-R:Fc polypeptide to BAFF is retained. Inother embodiments, the mutants encode a BAFF-R or BAFF-R:Fc polypeptidehaving amino acids that are not conserved between human and mouse BAFF-Rwhich have been changed to other amino acids. Preferably, at least onenonpolar amino acid is changed to a proline residue or an unchargedpolar amino acid.

Antisense

Another aspect of the invention pertains to isolated antisense nucleicacid molecules that are hybridizable to or complementary to the nucleicacid molecule comprising the nucleotide sequence of FIG. 2A, C, 3 or, orfragments, analogs or derivatives thereof. An “antisense” nucleic acidcomprises a nucleotide sequence that is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. In specific aspects, antisense nucleic acid molecules areprovided that comprise a sequence complementary to at least about 10,25, 50, 100, 250 or 500 nucleotides or an entire BAFF-R coding strand,or to only a portion thereof. Nucleic acid molecules encoding fragments,homologs, derivatives and analogs of a BAFF-R protein of any of FIG. 2A(SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6) or antisensenucleic acids complementary to a BAFF-R nucleic acid sequence of any ofFIG. 2A (SEQ ID NO:3), FIG. 2C(SEQ ID NO:4), FIG. 3 (SEQ ID NO:6) areadditionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encodingBAFF-R. The term “coding region” refers to the region of the nucleotidesequence comprising codons which are translated into amino acid residues(e.g., the protein coding region of human BAFF-R corresponds tonucleotides 13 to 568 of FIG. 2A (SEQ ID NO:3), or nucleotides 13 to 565of FIG. 2C (SEQ ID NO:4) or nucleotides 298 to 849 of FIG. 3 (SEQ IDNO:6)). In another embodiment, the antisense nucleic acid molecule isantisense to a “noncoding region” of the coding-strand of a nucleotidesequence encoding BAFF-R. The term “noncoding region” refers to 5′ and3′ sequences which flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding BAFF-R disclosed herein,antisense nucleic acids of the invention can be designed according tothe rules of Watson and Crick or Hoogsteen base pairing. The antisensenucleic acid molecule can be complimentary to the entire coding regionof BAFF-R mRNA, but more preferably is an oligonucleotide that isantisense to only a portion of the coding or noncoding region of BAFF-RmRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of BAFF-R mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid ofthe invention can be constructed using chemical synthesis or enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused.

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a BAFF-Rprotein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule that binds toDNA duplexes, through specific interactions in the major groove of thedouble helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies that bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of antisense molecules,vector constructs in which the antisense nucleic acid molecule is placedunder the control of a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an a-anomeric nucleic acid molecule. An a-anomeric nucleicacid molecule forms-specific double-stranded hybrids with complementaryRNA in which, contrary to the usual b-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucl. Acids Res. 15:6625-6641). Theantisense nucleic acid molecule can also comprise a2′-O-methylribonucleotide (Inoue et al. (1987) Nucl. Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

Ribozymes and PNA Moieties

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity that are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveBAFF-R mRNA transcripts to thereby inhibit translation of BAFF-R mRNA. Aribozyme having specificity for a BAFF-R-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a BAFF-R DNA disclosedherein (i.e., SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a BAFF-R-encoding mRNA. See, e.g.,Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.5,116,742. Alternatively, BAFF-R mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.

Alternatively, BAFF-R gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of theBAFF-R (e.g., the BAFF-R promoter and/or enhancers) to form triplehelical structures that prevent transcription of the BAFF-R gene intarget cells. See generally, Helene (1991) Anticancer Drug Des. 6:569-84; Helene et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher(1992) Bioassays 14:807-15.

In various embodiments, the nucleic acids of BAFF-R can be modified atthe base moiety, sugar moiety or phosphate backbone to improve, e.g.,the stability, hybridization, or solubility of the molecule. Forexample, the deoxyribose phosphate backbone of the nucleic acids can bemodified to generate peptide nucleic acids (see Hyrup et al. 1996)Bioorg. Med. Chem. 4:5-23). As used herein, the terms “peptide nucleicacids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, inwhich the deoxyribose phosphate backbone is replaced by a pseudopeptidebackbone and only the four natural nucleobases are retained. The neutralbackbone of PNAs has been shown to allow for specific hybridization toDNA and RNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup et al. (1996) Bioorg. Med. Chem. 4:5-23;Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs of BAFF-R can be used in therapeutic and diagnostic applications.For example, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs ofBAFF-R can also be used, e.g., in the analysis of single base pairmutations in a gene by, e.g., PNA directed PCR clamping; as artificialrestriction enzymes when used in combination with other enzymes, e.g.,S1 nucleases (Hyrup B. (1996) Bioorg. Med. Chem. 4:5-23); or as probesor primers for DNA sequence and hybridization (Hyrup et al. (1996),Bioorg. Med. Chem. 4:5-23; Perry-O'Keefe (1996) Proc. Natl. Acad. Sci.USA 93:14670-675).

In another embodiment, PNAs of BAFF-R can be modified, e.g., to enhancetheir stability or cellular uptake, by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of BAFF-R can be generated that maycombine the advantageous properties of PNA and DNA. Such chimeras allowDNA recognition enzymes, e.g., RNase H and DNA polymerases, to interactwith the DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation Hyrup (1996) Bioorg. Med.Chem. 4:5-23). The synthesis of PNA-DNA chimeras can be performed asdescribed in Hyrup (1996) Bioorg. Med. Chem. 4:5-23; and Finn et al.(1996) Nucl. Acids Res. 24:3357-63. For example, a DNA chain can besynthesized on a solid support using standard phosphoramidite couplingchemistry, and modified nucleoside analoǵs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNAand the 5′ end of DNA (Mag et al. (1989) Nucl. Acids Res. 17:5973-88).PNA monomers are then coupled in a stepwise manner to produce a chimericmolecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al. (1996)above). Alternatively, chimeric molecules can be synthesized with a 5′DNA segment and a 3′ PNA segment. See, Petersen et al. (1975) Bioorg.Med. Chem. Lett. 5:1119-11124.

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. W0 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. W0 89/10134). In addition, oligonucleotides can bemodified with hybridization triggered cleavage agents (see, e.g., Krolet al., (1988) BioTechniques 6:958-976) or intercalating agents (see,e.g., Zon, (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,a hybridization triggered cross-linking agent, a transport agent, ahybridization-triggered cleavage agent, etc.

BAFF-R Polypeptides

One aspect of the invention pertains to isolated BAFF-R proteins, andbiologically active portions thereof, or derivatives, fragments, analogsor homologs thereof. Also provided are polypeptide fragments suitablefor use as immunogens to raise anti-BAFF-R antibodies. In oneembodiment, native BAFF-R proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, BAFF-R proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a BAFF-R protein or polypeptide can be synthesizedchemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theBAFF-R protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofBAFF-R protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of BAFF-R protein having lessthan about 30% (by dry weight) of non-BAFF-R protein (also referred toherein as a “contaminating protein”), more preferably less than about20% of non-BAFF-R protein, still more preferably less than about 10% ofnon-BAFF-R protein, and most preferably less than about 5% non-BAFF-Rprotein. When the BAFF-R protein or biologically active portion thereofis recombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of BAFF-R protein in which the proteinis separated from chemical precursors or other chemicals that areinvolved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of BAFF-R protein having less than about 30% (bydry weight) of chemical precursors or non-BAFF-R chemicals, morepreferably less than about 20% chemical precursors or non-BAFF-Rchemicals, still more preferably less than about 10% chemical precursorsor non-BAFF-R chemicals, and most preferably less than about 5% chemicalprecursors or non-BAFF-R chemicals.

Biologically active portions of a BAFF-R protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the BAFF-R protein, e.g., the amino acidsequence shown in SEQ ID NO:5 that include fewer amino acids than thefull length BAFF-R proteins, and exhibit at least one activity of aBAFF-R protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the BAFF-R protein. Abiologically active portion of a BAFF-R protein can be a polypeptidewhich is, for example, 10, 25, 50, 100 or more amino acids in length.

A biologically active portion of a BAFF-R protein of the presentinvention may contain at least one of the above-identified domainsconserved between the BAFF-R proteins. An alternative biologicallyactive portion of a BAFF-R protein may contain at least two of theabove-identified domains. Another biologically active portion of aBAFF-R protein may contain at least three of the above-identifieddomains. Yet another biologically active portion of a BAFF-R protein ofthe present invention may contain at least four of the above-identifieddomains.

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a nativeBAFF-R protein.

In an embodiment, the BAFF-R protein has an amino acid sequence shown inFIG. 2D (SEQ ID NO:5). In other embodiments, the BAFF-R protein issubstantially homologous to FIG. 2D (SEQ ID NO:5) and retains thefunctional activity of the protein of FIG. 2D (SEQ ID NO:5), yet differsin amino acid sequence due to natural allelic variation or mutagenesis,as described in detail below. Accordingly, in another embodiment, theBAFF-R protein is a protein that comprises an amino acid sequence atleast about 45% homologous to the amino acid sequence of FIG. 2D (SEQ IDNO:5) and retains the functional activity of the BAFF-R proteins of FIG.2D (SEQ ID NO:5).

In some embodiments, the invention includes specific mutants ofBAFF-R:Fc polypeptide designed to alleviate aggregation of expressedprotein while maintaining BAFF binding activity. Such mutants, include,for example, clones encoding the amino acid sequences of JST661 (SEQ IDNO:17), JST662 (SEQ ID NO:18), JST663 (SEQ ID NO:19), JST673 (SEQ IDNO:20), JST674 (SEQ ID NO:21), JST675 (SEQ ID NO:22), JST672 (SEQ IDNO:23), JST676 (SEQ ID NO:24), JST671 (SEQ ID NO:25), JST677 (SEQ IDNO:26), and JST678 (SEQ ID NO:27). Other embodiments include mutantsencoding a BAFF-R or BAFF-R:Fc polypeptide that has similar aggregationcharacteristics to native human BAFF-R R or BAFF-R:Fc polypeptide, butalso bind BAFF, including, for example, sequences comprising the aminoacid sequences of JST659 (SEQ ID NO:15), JST660 (SEQ ID NO:16), JST664(SEQ ID NO:28), JST668 (SEQ ID NO:29), JST665 (SEQ ID NO:30), JST666(SEQ ID NO:31), and JST667 (SEQ ID NO:32). Other embodiments includemutants encoding a BAFF-R or BAFF-R:Fc polypeptide wherein conservedamino acids between human and mouse BAFF-R are changed to otherconserved amino acids and wherein the binding activity of BAFF-R orBAFF-R:Fc polypeptide to BAFF is retained. In other embodiments, themutants encode a BAFF-R or BAFF-R:Fc polypeptide having amino acids thatare not conserved between human and mouse BAFF-R which have been changedto other amino acids. Preferably, nonpolar amino acids are mutated toproline or uncharged polar amino acids.

Determining Homology Between Two or More Sequences

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are homologous at that position(i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”).

The nucleic acid sequence homology may be determined as the degree ofidentity between two sequences. The homology may be determined usingcomputer programs known in the art, such as GAP software provided in theGCG program package. See Needleman and Wunsch (1970) J. Mol. Biol.48:443-453. Using GCG GAP software with the following settings fornucleic acid sequence comparison: GAP creation penalty of 5.0 and GAPextension penalty of 0.3, the coding region of the analogous nucleicacid sequences referred to above exhibits a degree of identitypreferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, withthe CDS (encoding) part of the DNA sequence shown in FIG. 2A (SEQ IDNO:3), FIG. 2C (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6).

The term “sequence identity” refers to the degree to which twopolynucleotide or polypeptide sequences are identical on aresidue-by-residue basis over a particular region of comparison. Theterm “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over that region of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I, in the case of nucleic acids) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the region ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The term “substantialidentity” as used herein denotes a characteristic of a polynucleotidesequence, wherein the polynucleotide comprises a sequence that has atleast 80 percent sequence identity, preferably at least 85 percentidentity and often 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison region.

Chimeric and Fusion Proteins

The invention also provides BAFF-R chimeric or fusion proteins. As usedherein, a BAFF-R “chimeric protein” or “fusion protein” comprises aBAFF-R polypeptide operatively linked to a non-BAFF-R polypeptide. A“BAFF-R polypeptide” refers to a polypeptide having an amino acidsequence corresponding to BAFF-R, whereas a “non-BAFF-R polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein that is not substantially homologous to the BAFF-R protein,e.g., a protein that is different from the BAFF-R protein and that isderived from the same or a different organism. Within a BAFF-R fusionprotein the BAFF-R polypeptide can correspond to all or a portion of aBAFF-R protein. In one embodiment, a BAFF-R fusion protein comprises atleast one biologically active portion of a BAFF-R protein. In anotherembodiment, a BAFF-R fusion protein comprises at least two biologicallyactive portions of a BAFF-R protein. In yet another embodiment, a BAFF-Rfusion protein comprises at least three biologically active portions ofa BAFF-R protein. Within the fusion protein, the term “operativelylinked” is intended to indicate that the BAFF-R polypeptide and thenon-BAFF-R polypeptide are fused in-frame to each other. The non-BAFF-Rpolypeptide can be fused to the N-terminus or C-terminus of the BAFF-Rpolypeptide. The non-BAFF-R polypeptide may be, for example, the Fcportion of an antibody. This may be operatively joined to either theN-terminus or the C-terminus of the BAFF-R polypeptide. Fc-targetprotein fusions have been described in Lo et al. (1998) ProteinEngineering 11:495-500, and U.S. Pat. Nos. 5,541,087 and 5,726,044. Thedisclosures of which are herein incorporated by reference.

For example, in one embodiment a BAFF-R fusion protein comprises aBAFF-R domain operably linked to the extracellular domain of a secondprotein. Such fusion proteins can be further utilized in screeningassays for compounds which modulate BAFF-R activity (such assays aredescribed in detail below).

In yet another embodiment, the fusion protein is a GST-BAFF-R fusionprotein in which the BAFF-R sequences are fused to the C-terminus of theGST (i.e., glutathione S-transferase) sequences. Such fusion proteinscan facilitate the purification of recombinant BAFF-R.

In another embodiment, the fusion protein is a BAFF-R protein containinga heterologous signal sequence at its N-terminus. For example, sinceBAFF-R does not contain its own signal sequence, a heterologous signalsequence must be fused to the 5′ end of the BAFF-R coding sequence forefficient secretion of the BAFF-R fusion protein. Expression and/orsecretion of BAFF-R can be increased through use of differentheterologous signal sequences.

In yet another embodiment, the fusion protein is a BAFF-R-immunoglobulinfusion protein in which the BAFF-R sequences comprising one or moredomains are fused to sequences derived from a member of theimmunoglobulin protein family. The BAFF-R-immunoglobulin fusion proteinsof the invention can be incorporated into pharmaceutical compositionsand administered to a subject to inhibit an interaction between a BAFF-Rligand and a BAFF-R protein on the surface of a cell, to therebysuppress BAFF-R-mediated signal transduction in vivo. TheBAFF-R-immunoglobulin fusion proteins can be used to affect thebioavailability of a BAFF-R cognate ligand. Inhibition of the BAFF-Rligand/BAFF-R interaction may be useful therapeutically for both thetreatment of proliferative and differentiative disorders, as well asmodulating (e.g. promoting or inhibiting) cell survival. Moreover, theBAFF-R-immunoglobulin fusion proteins of the invention can be used asimmunogens to produce anti-BAFF-R antibodies in a subject, to purifyBAFF-R ligands, and in screening assays to identify molecules thatinhibit the interaction of BAFF-R with a BAFF-R ligand.

A BAFF-R chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Ausubel et al. Eds. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A BAFF-R-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to theBAFF-R protein.

In a preferred embodiment, the BAFF-R fusion is provided by the nucleicacid (SEQ ID NO:11) and amino acid (SEQ ID NO:12) sequences of FIG. 9.

BAFF-R Agonists and Antagonists

The present invention also pertains to variants of the BAFF-R proteinsthat function as either BAFF-R agonists (mimetics) or as BAFF-Rantagonists. Variants of the BAFF-R protein can be generated bymutagenesis, e.g., discrete point mutation or truncation of the BAFF-Rprotein. An agonist of the BAFF-R protein can retain substantially thesame, or a subset of, the biological activities of the naturallyoccurring form of the BAFF-R protein. An antagonist of the BAFF-Rprotein can inhibit one or more of the activities of the naturallyoccurring form of the BAFF-R protein by, for example, competitivelybinding to a downstream or upstream member of a cellular signalingcascade which includes the BAFF-R protein. Thus, specific biologicaleffects can be elicited by treatment with a variant of limited function.In one embodiment, treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein has fewer side effects in a subject relative to treatment withthe naturally occurring form of the BAFF-R proteins.

Variants of the BAFF-R protein that function as either BAFF-R agonists(mimetics) or as BAFF-R antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theBAFF-R protein for BAFF-R protein agonist or antagonist activity. In oneembodiment, a variegated library of BAFF-R variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of BAFF-R variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential BAFF-R sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of BAFF-R sequences therein. There are avariety of methods which can be used to produce libraries of potentialBAFF-R variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential BAFF-R sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang (1983)Tetrahedron 39:3; Itakura et al. (1984) Ann. Rev. Biochem. 53:323;Itakura et al. (1977) Science 198:1056-1063; Ike et al. (1983) Nucl.Acids Res. 11:477-488.

Polypeptide Libraries

In addition, libraries of fragments of the BAFF-R protein codingsequence can be used to generate a variegated population of BAFF-Rfragments for screening and subsequent selection of variants of a BAFF-Rprotein. In one embodiment, a library of coding sequence fragments canbe generated by treating a double stranded PCR fragment of aBAFF-R-coding sequence with a nuclease under conditions wherein nickingoccurs only about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA that can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal and internal fragments of various sizes of the BAFF-Rprotein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of BAFF-R proteins. The mostwidely used techniques, which are amenable to high throughput analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recrusive ensemble mutagenesis (REM), a newtechnique that enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify BAFF-R variants (Arkin and Yourvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6:327-331).

Anti-BAFF-R Antibodies

An isolated BAFF-R protein, or a portion or fragment thereof, can beused as an immunogen to generate antibodies that bind BAFF-R usingstandard techniques for polyclonal and monoclonal antibody preparation.The full-length BAFF-R protein can be used or, alternatively, theinvention provides antigenic peptide fragments of BAFF-R for use asimmunogens. The antigenic peptide of BAFF-R comprises at least 8 aminoacid residues of the amino acid sequence shown in FIG. 2D (SEQ ID NO:5)and encompasses an epitope of BAFF-R such that an antibody raisedagainst the peptide forms a specific immune complex with BAFF-R.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues. Preferred epitopes encompassed by the antigenicpeptide are regions of BAFF-R that are located on the surface of theprotein, e.g., hydrophilic regions.

As disclosed herein, BAFF-R protein sequence of FIG. 2D (SEQ ID NO:5),or derivatives, fragments, analogs or homologs thereof, may be utilizedas immunogens in the generation of antibodies thatimmunospecifically-bind these protein components. The term “antibody” asused herein refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site that specifically binds (immunoreactswith) an antigen, such as BAFF-R. Such antibodies include, but are notlimited to, polyclonal, monoclonal, chimeric, single chain, Fab andF(ab′)₂ fragments, and an Fab expression library. In a specificembodiment, antibodies to human BAFF-R proteins are disclosed. Variousprocedures known within the art may be used for the production ofpolyclonal or monoclonal antibodies to a BAFF-R protein sequence of FIG.2D (SEQ ID NO:5) or derivative, fragment, analog or homolog thereof.Some of these proteins are discussed below.

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byinjection with the native protein, or a synthetic variant thereof, or aderivative of the foregoing. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed BAFF-R protein or achemically synthesized BAFF-R polypeptide. The preparation can furtherinclude an adjuvant. Various adjuvants used to increase theimmunological response include, but are not limited to, Freund's(complete and incomplete), mineral gels (e.g. aluminum hydroxide),surface active substances (e.g., lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, dinitrophenol, etc.), humanadjuvants such as Bacille Calmette-Guerin (BCG) and Corynebacteriumparvum, or similar immunostimulatory agents. If desired, the antibodymolecules directed against BAFF-R can be isolated from the mammal (e.g.,from the blood) and further purified by well-known techniques, such asprotein A chromatography to obtain the IgG fraction.

The term “monoclonal antibody” or “monoclonal antibody composition”, asused herein, refers to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of BAFF-R. A monoclonal antibody compositionthus typically displays a single binding affinity for a particularBAFF-R protein with which it immunoreacts. For preparation of monoclonalantibodies directed towards a particular BAFF-R protein, or derivatives,fragments, analogs or homologs thereof, any technique that provides forthe production of antibody molecules by continuous cell line culture maybe utilized. Such techniques include, but are not limited to, thehybridoma technique (see Kohler & Milstein, (1975) Nature 256:495-497);the trioma technique; the human B-cell hybridoma technique (see Kozboret al. (1983) Immunol. Today 4:72) and the EBV hybridoma technique toproduce human monoclonal antibodies (see Cole, et al. in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., 1985, pp. 77-96).Human monoclonal antibodies may be utilized in the practice of thepresent invention and may be produced by using human hybridomas (seeCote et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole etal. in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc.,1985 pp. 77-96).

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to a BAFF-R protein (see e.g., U.S.Pat. No. 4,946,778). In addition, methods can be adapted for theconstruction of Fab expression libraries (see e.g., Huse et al. (1989)Science 246:1275-1281) to allow rapid and effective identification ofmonoclonal Fab fragments with the desired specificity for a BAFF-Rprotein or derivatives, fragments, analogs or homologs thereof.Non-human antibodies can be “humanized” by techniques well known in theart. See e.g., U.S. Pat. No. 5,225,539. Antibody fragments that containthe idiotypes to a BAFF-R protein may be produced by techniques known inthe art including, but not limited to: (i) an F(ab′)₂ fragment producedby pepsin digestion of an antibody molecule; (ii) an Fab fragmentgenerated by reducing the disulfide bridges of an F(ab′)₂ fragment;(iii) an Fab fragment generated by the treatment of the antibodymolecule with papain and a reducing agent and (iv) Fv fragments.

Additionally, recombinant anti-BAFF-R antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art for example using methods described in PCTInternational Application No. PCT/US86/02269; European PatentApplication No. 184,187; European Patent Application No. 171,496;European Patent Application No. 173,494; PCT International PublicationNo. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent ApplicationNo. 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; Shaw et. al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler etal. (1988) J. Immunol. 141:4053-4060.

In one embodiment, methods for the screening of antibodies that possessthe desired specificity include, but are not limited to, enzyme-linkedimmunosorbent assay (ELISA) and other immunologically-mediatedtechniques known within the art. In a specific embodiment, selection ofantibodies that are specific to a particular domain of a BAFF-R proteinis facilitated by generation of hybridomas that bind to the fragment ofa BAFF-R protein possessing such a domain. Antibodies that are specificfor one or more domains within a BAFF-R protein, e.g., domains spanningthe above-identified conserved regions of BAFF-R family proteins, orderivatives, fragments, analogs or homologs thereof, are also providedherein.

Anti-BAFF-R antibodies may be used in methods known within the artrelating to the localization and/or quantitation of a BAFF-R protein(e.g., for use in measuring levels of the BAFF-R protein withinappropriate physiological samples, for use in diagnostic methods, foruse in imaging the protein, and the like). In a given embodiment,antibodies for BAFF-R proteins, or derivatives, fragments, analogs orhomologs thereof, that contain the antibody derived binding domain, areutilized as pharmacologically-active compounds (hereinafter“Therapeutics”).

An anti-BAFF-R antibody (e.g., monoclonal antibody) can be used toisolate BAFF-R by standard techniques, such as affinity chromatographyor immunoprecipitation. An anti-BAFF-R antibody can facilitate thepurification of natural BAFF-R from cells and of recombinantly producedBAFF-R expressed in host cells. Moreover, an anti-BAFF-R antibody can beused to detect BAFF-R protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the BAFF-R protein. Anti-BAFF-R antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, B-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

BAFF-R Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding BAFF-R protein,or derivatives, fragments, analogs or homologs thereof. As used herein,the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid,” which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors.” In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively lied so thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel “Gene Expression Technology” METHODSIN ENZYMOLOGY 185, Academic Press, San Diego, Calif., 1990. Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., BAFF-R proteins, mutant formsof BAFF-R, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of BAFF-R in prokaryotic or eukaryotic cells. For example,BAFF-R can be expressed in bacterial cells such as Escherichia coli,insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,“Gene Expression Technology” METHODS IN ENZYMOLOGY 185, Academic Press,San Diego, Calif., 1990. Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: (1) to increase expression ofrecombinant protein; (2) to increase the solubility of the recombinantprotein; and (3) to aid in the purification of the recombinant proteinby acting as a ligand in affinity purification. Often, in fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fission protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., “Gene Expression Technology” METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif., 1990, pp. 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacterium with an impaired capacity toproteolytically cleave the recombinant protein. See, Gottesman, “GeneExpression Technology” METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif., 1990, pp. 119-128. Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al., (1992) NucleicAcids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the BAFF-R expression vector is a yeastexpression vector. Examples of vectors for expression in yeast (e.g.,Saccharomyces cerivisae) include pYepSec1 (Baldari, et al., (1987) EMBOJ. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2(InvitrogenCorporation, San Diego, Calif.), and for P. pastoris include pPIC familyof vectors (Invitrogen Corp, San Diego, Calif.).

Alternatively, BAFF-R can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., SF9 cells) include the pAcseries (Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVLseries (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature329:840-842) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). Whenused in mammalian cells, the expression vector's control functions areoften provided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 ofSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2ND ED., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter, U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine box promoters (Kessel and Gruss (1990)Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman(1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to BAFF-R mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen that direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen that directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub et al.(1986) “Antisense RNA as a molecular tool for genetic analysis,”Reviews—Trends in Genetics, 1(1).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,BAFF-R protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. MOLECULARCLONING: A LABORATORY MANUAL 2ND ED., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding BAFF-R or can be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) BAFF-R protein.Accordingly, the invention further provides methods for producing BAFF-Rprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding BAFF-R has been introduced) in asuitable medium such that BAFF-R protein is produced. In anotherembodiment, the method further comprises isolating BAFF-R from themedium or the host cell.

Transgenic Animals

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichBAFF-R-coding sequences have been introduced. Such host cells can thenbe used to create non-human transgenic animals in which exogenous BAFF-Rsequences have been introduced into their genome or homologousrecombinant animals in which endogenous BAFF-R sequences have beenaltered. Such animals are useful for studying the function and/oractivity of BAFF-R and for identifying and/or evaluating modulators ofBAFF-R activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA that is integrated into the genome of a cellfrom which a transgenic animal develops and that remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, a “homologous recombinant animal” is a non-humananimal, preferably a mammal, more preferably a mouse, in which anendogenous BAFF-R gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

A transgenic animal of the invention can be created by introducingBAFF-R-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The humanBAFF-R DNA sequence of FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4),FIG. 3 (SEQ ID NO:6) can be introduced as a transgene into the genome ofa non-human animal. Alternatively, a nonhuman homologue of the humanBAFF-R gene, such as a mouse BAFF-R gene (FIG. 4A) (SEQ ID NO:8), can beisolated based on hybridization to the human BAFF-R cDNA (describedfurther above) and used as a transgene. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably lied to theBAFF-R transgene to direct expression of BAFF-R protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan in MANIPULATING THEMOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1986. Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the BAFF-R transgene in its genome and/or expression ofBAFF-R mRNA in tissues or cells of the animals. A transgenic founderanimal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encodingBAFF-R can further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a BAFF-R gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the BAFF-R gene. The BAFF-R gene can be a humangene (e.g., FIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), FIG. 3 (SEQ IDNO:6)), but more preferably; is a non-human homologue of a human BAFF-Rgene. For example, a mouse homologue (FIG. 4 a) of human BAFF-R gene ofFIG. 2A (SEQ ID NO:3), FIG. 2C (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6) canbe used to construct a homologous recombination vector suitable foraltering an endogenous BAFF-R gene in the mouse genome. In oneembodiment, the vector is designed such that, upon homologousrecombination, the endogenous BAFF-R gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as a“knock out” vector).

Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous BAFF-R gene is mutated or otherwisealtered but still encodes functional protein (e.g., the upstreamregulatory region can be altered to thereby alter the expression of theendogenous BAFF-R protein). In the homologous recombination vector, thealtered portion of the BAFF-R gene is flanked at its 5′ and 3′ ends byadditional nucleic acid of the BAFF-R gene to allow for homologousrecombination to occur between the exogenous BAFF-R gene carried by thevector and an endogenous BAFF-R gene in an embryonic stem cell. Theadditional flanking BAFF-R nucleic acid is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector. See e.g., Thomas et al (1987) Cell 51:503 for adescription of homologous recombination vectors. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced BAFF-R gene has homologouslyrecombined with the endogenous BAFF-R gene are selected (see e.g., Li etal. (1992) Cell 69:915).

The selected cells are then injected into a blastocyst of an animal(e.g., a mouse) to form aggregation chimeras. See e.g., Bradley, inTERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH,Robertson, Ed. IRL, Oxford, 1987, pp. 113-152. A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term. Progeny harboring the homologouslyrecombined DNA in their germ cells can be used to breed animals in whichall cells of the animal contain the homologously recombined DNA bygermline transmission of the transgene. Methods for constructinghomologous recombination vectors and homologous recombinant animals aredescribed further in Bradley (1991) Curr. Opin. Biotechnol. 2:823-829;PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO92/0968; and WO 93/04169.

In another embodiment, transgenic non-humans animals can be producedthat contain selected systems that allow for regulated expression of thetransgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of S. cerevisae (O'Gorman et al. (1991) Science251:1351-1355. If a cre/loxP recombinase system is used to regulateexpression of the transgene, animals containing transgenes encoding boththe Cre recombinase and a selected protein are required. Such animalscan be provided through the construction of “double” transgenic animals,e.g., by mating two transgenic animals, one containing a transgeneencoding a selected protein and the other containing a transgeneencoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G₀ phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyte and then transferred to pseudopregnant female foster animal.The offspring borne of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

Pharmaceutical Compositions

The BAFF-R nucleic acid molecules, BAFF-R proteins, and anti-BAFF-Rantibodies (also referred to herein as “active compounds”) of theinvention, and derivatives, fragments, analogs and homologs thereof, canbe incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, finger's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a BAFF-R protein or anti-BAFF-R antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by any of a number of routes, e.g., as described in U.S.Pat. No. 5,703,055. Delivery can thus also include, e.g., intravenousinjection, local administration (see U.S. Pat. No. 5,328,470) orstereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad.Sci. 91:3054-3057). The pharmaceutical preparation of the gene therapyvector can include the gene therapy vector in an acceptable diluent, orcan comprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods:(a) screening assays; (b) detection assays (e.g., chromosomal mapping,tissue typing, forensic biology), (c) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and (d) methods of treatment (e.g., therapeutic andprophylactic). As described herein, in one embodiment, a BAFF-R proteinof the invention has the ability to bind BAFF.

The isolated nucleic acid molecules of the invention can be used toexpress BAFF-R protein (e.g., via a recombinant expression vector in ahost cell in gene therapy applications), to detect BAFF-R mRNA (e.g., ina biological sample) or a genetic lesion in a BAFF-R gene, and tomodulate BAFF and/or BAFF-R activity, as described further below. Inaddition, the BAFF-R proteins can be used to screen drugs or compoundsthat modulate the BAFF-R activity or expression as well as to treatdisorders characterized by insufficient or excessive production of BAFFand/or BAFF-R protein; or production of BAFF-R protein forms that havedecreased or aberrant activity compared to BAFF-R wild type protein. Inaddition, the anti-BAFF-R antibodies of the invention can be used todetect and isolate BAFF-R proteins and modulate BAFF and/or BAFF-Ractivity.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g. peptides, peptidomimetics, small molecules or other drugs)that bind to BAFF-R proteins or have a stimulatory or inhibitory effecton, for example, BAFF-R expression or BAFF-R activity.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of a BAFF-Rprotein or polypeptide or biologically active portion thereof. The testcompounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam (1997) Anticancer Drug Des.12:145-167).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909-6013; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422-11426; Zuckermann et al. (1994) J. Med. Chem. 37:2678-2685; Choet al. (1993) Science 261:1303; Carrel et al. (1994) Angew Chem. Int.Ed. Engl. 33:2059; Carell et al. (1994) Angew Chem. Int. Ed. Engl.33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233-1251.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), on chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) oron phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990)Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner U.S. Pat.No. 5,223,409).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a membrane-bound form of BAFF-R protein, or a biologicallyactive portion thereof, on the cell surface is contacted with a testcompound and the ability of the test compound to bind to a BAFF-Rprotein determined. The cell, for example, can of mammalian origin or ayeast cell. Determining the ability of the test compound to bind to theBAFF-R protein can be accomplished, for example, by coupling the testcompound with a radioisotope or enzymatic label such that binding of thetest compound to the BAFF-R protein or biologically active portionthereof can be determined by detecting the labeled compound in acomplex. For example, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemission or by scintillation counting.Alternatively, test compounds can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product. In one embodiment, the assay comprisescontacting a cell which expresses a membrane-bound form of BAFF-Rprotein, or a biologically active portion thereof, on the cell surfacewith a known compound which binds BAFF-R to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a BAFF-R protein, whereindetermining the ability of the test compound to interact with a BAFF-Rprotein comprises determining the ability of the test compound topreferentially bind to BAFF-R or a biologically active portion thereofas compared to the known compound.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of BAFF-R protein, ora biologically active portion thereof, on the cell surface with a testcompound and determining the ability of the test compound to modulate(e.g., stimulate or inhibit) the activity of the BAFF-R protein orbiologically active portion thereof. Determining the ability of the testcompound to modulate the activity of BAFF-R or a biologically activeportion thereof can be accomplished, for example, by determining theability of the BAFF-R protein to bind to or interact with a BAFF-Rtarget molecule. As used herein, a “target molecule” is a molecule withwhich a BAFF-R protein binds or interacts in nature, for example, amolecule on the surface of a cell which expresses a BAFF-R protein, amolecule on the surface of a second cell, a molecule in theextracellular milieu, a molecule associated with the internal surface ofa cell membrane or a cytoplasmic molecule. A BAFF-R target molecule canbe a non-BAFF-R molecule or a BAFF-R protein or polypeptide of thepresent invention. In one embodiment, a BAFF-R target molecule is acomponent of a signal transduction pathway that facilitates transductionof an extracellular signal (e.g., a signal generated by binding of acompound to a membrane-bound BAFF-R molecule) through the cell membraneand into the cell. The target, for example, can be a secondintercellular protein that has catalytic activity or a protein thatfacilitates the association of downstream signaling molecules withBAFF-R.

Determining the ability of the BAFF-R protein to bind to or interactwith a BAFF-R target molecule can be accomplished by one of the methodsdescribed above for determining direct binding. In one embodiment,determining the ability of the BAFF-R protein to bind to or interactwith a BAFF-R target molecule can be accomplished by determining theactivity of the target molecule. For example, the activity of the targetmolecule can be determined by detecting induction of a cellular secondmessenger of the target (i.e. intracellular Ca²⁺, diacylglycerol, IP3,etc.), detecting catalytic/enzymatic activity of the target anappropriate substrate, detecting the induction of a reporter gene(comprising a BAFF-R-responsive regulatory element operatively linked toa nucleic acid encoding a detectable marker, e.g., luciferase), ordetecting a cellular response, for example, cell survival, cellulardifferentiation, or cell proliferation.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a BAFF-R protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to bind to the BAFF-R protein or biologicallyactive portion thereof. Binding of the test compound to the BAFF-Rprotein can be determined either directly or indirectly as describedabove. In one embodiment, the assay comprises contacting the BAFF-Rprotein or biologically active portion thereof with a known compoundwhich binds BAFF-R to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with a BAFF-R protein, wherein determining theability of the test compound to interact with a BAFF-R protein comprisesdetermining the ability of the test compound to preferentially bind toBAFF-R or biologically active portion thereof as compared to the knowncompound.

In another embodiment, an assay is a cell-free assay comprisingcontacting BAFF-R protein or biologically active portion thereof with atest compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the BAFF-R proteinor biologically active portion thereof. Determining the ability of thetest compound to modulate the activity of BAFF-R can be accomplished,for example, by determining the ability of the BAFF-R protein to bind toa BAFF-R target molecule by one of the methods described above fordetermining direct binding. In an alternative embodiment, determiningthe ability of the test compound to modulate the activity of BAFF-R canbe accomplished by determining the ability of the BAFF-R protein furthermodulate a BAFF-R target molecule. For example, the catalytic/enzymaticactivity of the target molecule on an appropriate substrate can bedetermined as previously described.

In yet another embodiment, the cell-free assay comprises contacting theBAFF-R protein or biologically active portion thereof with a knowncompound which binds BAFF-R to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with a BAFF-R protein, wherein determining theability of the test compound to interact with a BAFF-R protein comprisesdetermining the ability of the BAFF-R protein to preferentially bind toor modulate the activity of a BAFF-R target molecule.

The cell-free assays of the present invention are amenable to use ofboth the soluble form or the membrane-bound form of BAFF-R. In the caseof cell-free assays comprising the membrane-bound form of BAFF-R, it maybe desirable to utilize a solubilizing agent such that themembrane-bound form of BAFF-R is maintained in solution. Examples ofsuch solubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS),3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either BAFF-R or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to BAFF-R, or interaction of BAFF-Rwith a target molecule in the presence and absence of a candidatecompound, can be accomplished in any vessel suitable for containing thereactants. Examples of such vessels include microtiter plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided that adds a domain that allows one or both of theproteins to be bound to a matrix. For example, GST-BAFF-R fusionproteins or GST-target fusion proteins can be adsorbed onto glutathionesepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathionederivatized microtiter plates, that are then combined with the testcompound or the test compound and either the non-adsorbed target proteinor BAFF-R protein, and the mixture is incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly, forexample, as described above. Alternatively, the complexes can bedissociated from the matrix, and the level of BAFF-R binding or activitydetermined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either BAFF-R orits target molecule can be immobilized utilizing conjugation of biotinand streptavidin. Biotinylated BAFF-R or target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive with BAFF-Ror target molecules, but which do not interfere with binding of theBAFF-R protein to its target molecule, can be derivatized to the wellsof the plate, and unbound target or BAFF-R trapped in the wells byantibody conjugation. Methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the BAFF-Ror target molecule, as well as enzyme-linked assays that rely ondetecting an enzymatic activity associated with the BAFF-R or targetmolecule.

In another embodiment, modulators of BAFF-R expression are identified ina method wherein a cell is contacted with a candidate compound and theexpression of BAFF-R mRNA or protein in the cell is determined. Thelevel of expression of BAFF-R mRNA or protein in the presence of thecandidate compound is compared to the level of expression of BAFF-R mRNAor protein in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of BAFF-R expressionbased on this comparison. For example, when expression of BAFF-R mRNA orprotein is greater (statistically significantly greater) in the presenceof the candidate compound than in its absence, the candidate compound isidentified as a stimulator of BAFF-R mRNA or protein expression.Alternatively, when expression of BAFF-R mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of BAFF-R mRNA or protein expression. The level of BAFF-R mRNAor protein expression in the cells can be determined by methodsdescribed herein for detecting BAFF-R mRNA or protein.

In yet another aspect of the invention, the BAFF-R proteins can be usedas “bait proteins” in a two-hybrid assay or three hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO 94/10300), to identify other proteins thatbind to or interact with BAFF-R (“BAFF-R-binding proteins” or“BAFF-R-bp”) and modulate BAFF-R activity. Such BAFF-R-binding proteinsare also likely to be involved in the propagation of signals by theBAFF-R proteins as, for example, upstream or downstream elements of theBAFF-R pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for BAFF-R is fused toa gene encoding the DNA binding domain of a known transcription factor(e.g., GAL-4). In the other construct, a DNA sequence, from a library ofDNA sequences, that encodes an unidentified protein (“prey” or “sample”)is fused to a gene that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract, in vivo, forming a BAFF-R-dependent complex, the DNA-bindingand activation domains of the transcription factor are brought intoclose proximity. This proximity allows transcription of a reporter gene(e.g., LacZ) that is operably linked to a transcriptional regulatorysite responsive to the transcription factor. Expression of the reportergene can be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genethat encodes the protein which interacts with BAFF-R.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the BAFF-R, sequences, described herein, can beused to map the location of the BAFF-R genes, respectively, on achromosome. The mapping of the BAFF-R sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

Briefly, BAFF-R genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the BAFF-R sequences.Computer analysis of the BAFF-R, sequences can be used to rapidly selectprimers that do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers can then be usedfor PCR screening of somatic cell hybrids containing individualchromosomes of a given species. Only those hybrids containing thespecies-specific gene corresponding to the BAFF-R sequences will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the BAFF-Rsequences to design oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical likecolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases, willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al. HUMAN CHROMOSOMES: A MANUAL OF BASICTECHNIQUES, Pergamon Press, N.Y., 1988.

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marling multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in McKusick,MENDELIAN INHERITANCE IN MAN, available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddisease, mapped to the same chromosomal region, can then be identifiedthrough linkage analysis (co-inheritance of physically adjacent genes),described in, for example, Egeland et al. (1987) Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the BAFF-R gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

Tissue Typing

The BAFF-R sequences of the present invention can also be used toidentify individuals from minute biological samples. In this technique,an individual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentification. The sequences of the present invention are useful asadditional DNA markers for RFLP (“restriction fragment lengthpolymorphisms,” described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique that determines the actual base-by-baseDNA sequence of selected portions of an individual's genome. Thus, theBAFF-R sequences described herein can be used to prepare two PCR primersfrom the 5′ and 3′ ends of the sequences. These primers can then be usedto amplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The BAFF-R sequences of the invention uniquely represent portions of thehuman genome. Allelic variation occurs to some degree in the codingregions of these sequences, and to a greater degree in the noncodingregions. It is estimated that allelic variation between individualhumans occurs with a frequency of about once per each 500 bases. Much ofthe allelic variation is due to single nucleotide polymorphisms (SNPs),which include restriction fragment length polymorphisms (RFLPs).

Each of the sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes. Because greater numbers of polymorphisms occurin the noncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of FIG. 1A (SEQ ID NO:1), FIG. 1B(SEQ ID NO:2), FIG. 2A (SEQ ID NO:3), FIG. 2B (SEQ ID NO:4), FIG. 3 (SEQID NO:6) can comfortably provide positive individual identification witha panel of perhaps 10 to 1,000 primers that each yield a noncodingamplified sequence of 100 bases. If predicted coding sequences, such asthose in FIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ IDNO:3), FIG. 2B (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6) are used, a moreappropriate number of primers for positive individual identificationwould be 500-2,000.

Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trials are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningBAFF-R protein and/or nucleic acid expression as well as BAFF-Ractivity, in the context of a biological sample (e.g., blood, serum,cells, tissue) to thereby determine whether an individual is afflictedwith a disease or disorder, or is at risk of developing a disorder,associated with aberrant BAFF-R expression or activity. The inventionalso provides for prognostic (or predictive) assays for determiningwhether an individual is at risk of developing a disorder associatedwith BAFF-R protein, nucleic acid expression or activity. For example,mutations in a BAFF-R gene can be assayed in a biological sample. Suchassays ran be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with BAFF-R protein, nucleic acidexpression or activity.

Another aspect of the invention provides methods for determining BAFF-Rprotein, nucleic acid expression or BAFF-R activity in an individual tothereby select appropriate therapeutic or prophylactic agents for thatindividual (referred to herein as “pharmacogenomics”). Pharmacogenomicsallows for the selection of agents (e.g., drugs) for therapeutic orprophylactic treatment of an individual based on the genotype of theindividual (e.g., the genotype of the individual examined to determinethe ability of the individual to respond to a particular agent.)

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs, compounds) on the expression or activity ofBAFF-R in clinical trials.

These and other agents are described in further detail in the followingsections.

Diagnostic Assays

An exemplary method for detecting the presence or absence of BAFF-R in abiological sample involves obtaining a biological sample from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting BAFF-R protein or nucleic acid (e.g., mRNA, genomicDNA) that encodes BAFF-R protein such that the presence of BAFF-R isdetected in the biological sample. An agent for detecting BAFF-R mRNA orgenomic DNA is a labeled nucleic acid probe capable of hybridizing toBAFF-R mRNA or genomic DNA. The nucleic acid probe can be, for example,a full-length BAFF-R nucleic acid, such as the nucleic acids of any ofFIG. 1A (SEQ ID NO:1), FIG. 1B (SEQ ID NO:2), FIG. 2A (SEQ ID NO:3),FIG. 2B (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6) or a portion thereof, suchas an oligonucleotide of at least 15, 30, 50, 100, 250 or 500nucleotides in length and sufficient to specifically hybridize understringent conditions to BAFF-R mRNA or genomic DNA. Other suitableprobes for use in the diagnostic assays of the invention are describedherein.

An agent for detecting BAFF-R protein is an antibody capable of bindingto BAFF-R protein, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g. Fab or F(ab′)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. That is, the detection method of the inventioncan be used to detect BAFF-R mRNA, protein, or genomic DNA in abiological sample in vitro as well as in vivo. For example, in vitrotechniques for detection of BAFF-R mRNA include Northern hybridizationsand in situ hybridizations. In vitro techniques for detection of BAFF-Rprotein include enzyme linked immunosorbent assays (ELISAs), Westernblots, immunoprecipitations and immunofluorescence. In vitro techniquesfor detection of BAFF-R genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of BAFF-R protein includeintroducing into a subject a labeled anti-BAFF-R antibody. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a peripheral blood leukocytesample isolated by conventional means from a subject.

In another embodiment, the methods flirter involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting BAFF-R protein, mRNA, orgenomic DNA, such that the presence of BAFF-R protein, mRNA or genomicDNA is detected in the biological sample, and comparing the presence ofBAFF-R protein, mRNA or genomic DNA in the control sample with thepresence of BAFF-R protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of BAFF-Rin a biological sample. For example, the kit can comprise: a labeledcompound or agent capable of detecting BAFF-R protein or mRNA in abiological sample; means for determining the amount of BAFF-R in thesample; and means for comparing the amount of BAFF-R in the sample witha standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect BAFF-R protein or nucleic acid.

Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant BAFF-R expression or activity. For example, theassays described herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with BAFF-R protein, nucleicacid expression or activity in, e.g., autoimmune conditions such asautoimmune hemolytic anemia and systemic lupus erythematosus.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing a disease or disorder. Thus,the present invention provides a method for identifying a disease ordisorder associated with aberrant BAFF-R expression or activity in whicha test sample is obtained from a subject and BAFF-R protein or nucleicacid (e.g., mRNA, genomic DNA) is detected, wherein the presence ofBAFF-R protein or nucleic acid is diagnostic for a subject having or atrisk of developing a disease or disorder associated with aberrant BAFF-Rexpression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., serum), cell sample, ortissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant BAFF-R expression or activity. For example,such methods can be used to determine whether a subject can beeffectively treated with an agent for a disorder. Thus, the presentinvention provides methods for determining whether a subject can beeffectively treated with an agent for a disorder associated withaberrant BAFF-R expression or activity in which a test sample isobtained and BAFF-R protein or nucleic acid is detected (e.g., whereinthe presence of BAFF-R protein or nucleic acid is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant BAFF-R expression or activity.)

The methods of the invention can also be used to detect genetic lesionsin a BAFF-R gene, thereby determining if a subject with the lesionedgene is at risk for, or suffers from, a tumorigenic or autoimmunedisorder. In various embodiments, the methods include detecting, in asample of cells from the subject, the presence or absence of a geneticlesion characterized by at least one of an alteration affecting theintegrity of a gene encoding a BAFF-R-protein, or the mis-expression ofthe BAFF-R gene. For example, such genetic lesions can be detected byascertaining the existence of at least one of (1) a deletion of one ormore nucleotides from a BAFF-R gene; (2) an addition of one or morenucleotides to a BAFF-R gene; (3) a substitution of one or morenucleotides of a BAFF-R gene, (4) a chromosomal rearrangement of aBAFF-R gene; (5) an alteration in the level of a messenger RNAtranscript of a BAFF-R gene, (6) aberrant modification of a BAFF-R gene,such as of the methylation pattern of the genomic DNA, (7) the presenceof a non-wild type splicing pattern of a messenger RNA transcript of aBAFF-R gene, (8) a non-wild type level of a BAFF-R-protein, (9) allelicloss of a BAFF-R gene, and (10) inappropriate post-translationalmodification of a BAFF-R-protein. As described herein, there are a largenumber of assay techniques known in the art which can be used fordetecting lesions in a BAFF-R gene. A preferred biological sample is aperipheral blood leukocyte sample isolated by conventional means from asubject. However, any biological sample containing nucleated cells maybe used, including, for example, buccal mucosal cells.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the BAFF-R-gene(see Abravaya et al. (1995) Nucl. Acids Res. 23:675-682). This methodcan include the steps of collecting a sample of cells from a patient,isolating nucleic acid (e.g., genomic, in RNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primersthat specifically hybridize to a BAFF-R gene under conditions such thathybridization and amplification of the BAFF-R gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) BioTechnology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a BAFF-R gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,493,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in BAFF-R can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al.(1996) Nature Med. 2:753-759). For example, genetic mutations in BAFF-Rcan be identified in two-dimensional arrays containing light-generatedDNA probes as described in Cronin et al. (1996) Human Mutation7:244-255. Briefly, a first hybridization array of probes can be used toscan through long stretches of DNA in a sample and control to identifybase changes between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe, arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the BAFF-R gene anddetect mutations by comparing the sequence of the sample BAFF-R with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxim andGilbert (1977) Proc. Natl. Acad. Sci. USA 74:560 or Sanger (1977) Proc.Natl. Acad. Sci. USA 74:5463. It is also contemplated that any of avariety of automated sequencing procedures can be utilized whenperforming the diagnostic assays (Naeve et al. (1995) Biotechniques19:448), including sequencing by mass spectrometry (see, e.g., PCTInternational Publ. No. WO 94/16101; Cohen et al. (1996)Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

Other methods for detecting mutations in the BAFF-R gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes of formed by hybridizing (labeled) RNA orDNA containing the wild-type BAFF-R sequence with potentially mutant RNAor DNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent that cleaves single-stranded regions of the duplexsuch as which will exist due to base pair mismatches between the controland sample strands. For instance, RNA/DNA duplexes can be treated withRNase and DNA/DNA hybrids treated with S1 nuclease to enzymaticallydigesting the mismatched regions. In other embodiments, either DNA/DNAor RNA/DNA duplexes can be treated with hydroxylamine or osmiumtetroxide and with piperidine in order to digest mismatched regions.After digestion of the mismatched regions, the resulting material isthen separated by size on denaturing polyacrylamide gels to determinethe site of mutation. See, for example, Cotton et al. (1988) Proc. Natl.Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol.217:286-295. In an embodiment, the control DNA or RNA can be labeled fordetection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in BAFF-R cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a BAFF-Rsequence, e.g., a wild-type BAFF-R sequence, is hybridized to a cDNA orother DNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, for example,U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in BAFF-R genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766, see also Cotton(1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl.9:73-79). Single-stranded DNA fragments of sample and control BAFF-Rnucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In one embodiment,the subject method utilizes heteroduplex analysis to separate doublestranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions thatpermit hybridization only if a perfect match is found (Saiki et al.(1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA86:6230). Such allele specific oligonucleotides are hybridized to PCRamplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology that depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucl. Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell. Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence, making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a BAFF-R gene.

Furthermore, any cell type or tissue, in which BAFF-R is expressed maybe utilized in the prognostic assays described herein. However, anybiological sample containing nucleated cells may be used, including, forexample, buccal mucosal cells.

Pharmacogenomics

Agents, or modulators that have a stimulatory or inhibitory effect onBAFF-R activity (e.g., BAFF-R gene expression), as identified by ascreening assay described herein can be administered to individuals totreat (prophylactically or therapeutically) disorders (e.g.,cancer-related or autoimmune disorders). In conjunction with suchtreatment, the pharmacogenomics (i.e., the study of the relationshipbetween an individual's genotype and that individual's response to aforeign compound or drug) of the individual may be considered.Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, thepharmacogenomics of the individual permits the selection of effectiveagents (e.g. drugs) for prophylactic or therapeutic treatments based ona consideration of the individual's genotype. Such pharmacogenomics canfurther be used to determine appropriate dosages and therapeuticregimens. Accordingly, the activity of BAFF-R protein, expression ofBAFF-R nucleic acid, or mutation content of BAFF-R genes in anindividual can be determined to thereby select appropriate agent(s) fortherapeutic or prophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See e.g., Eichelbaum (1996) Clin. Exp.Pharmacol. Physiol. 23:983-985 and Linder (1997) Clin. Chem. 43:254-266.In general, two types of pharmacogenetic conditions can bedifferentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase (G6PD) deficiency is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of BAFF-R protein, expression of BAFF-R nucleic acid,or mutation content of BAFF-R genes in an individual can be determinedto thereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual. In addition, pharmacogenetic studies can beused to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith a BAFF-R modulator, such as a modulator identified by one of theexemplary screening assays described herein.

Monitoring Clinical Efficacy

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of BAFF-R (e.g., the ability to modulate aberrantcell proliferation and/or differentiation) can be applied not only inbasic drug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase BAFF-R gene expression, protein levels, or upregulateBAFF-R activity, can be monitored in clinical trials of subjectsexhibiting decreased BAFF-R gene expression, protein levels, ordownregulated BAFF-R activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease BAFF-R geneexpression, protein levels, or downregulate BAFF-R activity, can bemonitored in clinical trials of subjects exhibiting increased BAFF-Rgene expression, protein levels, or upregulated BAFF-R activity. In suchclinical trials, the expression or activity of BAFF-R and, preferably,other genes that have been implicated in, for example, a disorder, canbe used as a “read out” or markers of the immune responsiveness of aparticular cell.

For example, genes, including BAFF-R, that are modulated in cells bytreatment with an agent (e.g., compound, drug or small molecule) thatmodulates BAFF-R activity (e.g., identified in a screening assay asdescribed herein) can be identified. Thus, to study the effect of agentson cellular proliferation disorders, for example, in a clinical trial,cells can be isolated and RNA prepared and analyzed for the levels ofexpression of BAFF-R and other genes implicated in the disorder. Thelevels of gene expression (i.e., a gene expression pattern) can bequantified by Northern blot analysis or RT-PCR, as described herein, oralternatively by measuring the amount of protein produced, by one of themethods as described herein, or by measuring the levels of activity ofBAFF-R or other genes. In this way, the gene expression pattern canserve as a marker, indicative of the physiological response of the cellsto the agent. Accordingly, this response state may be determined before,and at various points during, treatment of the individual with theagent.

In one embodiment, the invention provides a method for monitoring theeffectiveness of treatment of a subject with an agent (e.g., an agonist,antagonist, protein, peptide, peptidomimetic, nucleic acid, smallmolecule, or other drug candidate identified by the screening assaysdescribed herein) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a BAFF-R protein, mRNA,or genomic DNA in the preadministration sample; (iii) obtaining one ormore post-administration samples from the subject; (iv) detecting thelevel of expression or activity of the BAFF-R protein, mRNA, or genomicDNA in the post-administration samples; (v) comparing the level ofexpression or activity of the BAFF-R protein, mRNA, or genomic DNA inthe pre-administration sample with the BAFF-R protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of BAFF-R to higher levels than detected, i.e.,to increase the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of BAFF-R to lower levels than detected, i.e., to decrease theeffectiveness of the agent.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant BAFF-R expression oractivity.

Diseases and disorders that are characterized by increased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with therapeutics that antagonize(i.e., reduce or inhibit) activity. Therapeutics that antagonizeactivity may be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to, (i) aBAFF-R polypeptide, or analogs, derivatives, fragments or homologsthereof; (ii) antibodies to a BAFF-R peptide; (iii) nucleic acidsencoding a BAFF-R peptide; (iv) administration of antisense nucleic acidand nucleic acids that are “dysfunctional” (i.e., due to a heterologousinsertion within the coding sequences of coding sequences to a BAFF-Rpeptide) are utilized to “knockout” endogenous function of a BAFF-Rpeptide by homologous recombination (see, e.g., Capecchi (1989) Science244: 1288-1292); or (v) modulators (i.e., inhibitors, agonists andantagonists, including additional peptide mimetic of the invention orantibodies specific to a peptide of the invention) that alter theinteraction between a BAFF-R peptide and its binding partner.

Diseases and disorders that are characterized by decreased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with Therapeutics that increase(i.e., are agonists to) activity. Therapeutics that upregulate activitymay be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to, aBAFF-R peptide, or analogs, derivatives, fragments or homologs thereof;or an agonist that increases bioavailability.

Increased or decreased levels can be readily detected by quantifyingpeptide and/or RNA, by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying it in vitro for RNA or peptide levels,structure and/or activity of the expressed peptides (or mRNAs of aBAFF-R peptide). Methods that are well-known within the art include, butare not limited to, immunoassays (e.g., by Western blot analysis,immunoprecipitation followed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/orhybridization assays to detect expression of mRNAs (e.g., Northernassays, dot blots, in situ hybridization, etc.).

In one aspect, the invention provides a method for preventing, in asubject, a disease or condition associated with an aberrant BAFF-Rexpression or activity, by administering to the subject an agent thatmodulates BAFF-R expression or at least one BAFF-R activity. Subjects atrisk for a disease that is caused or contributed to by aberrant BAFF-Rexpression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the BAFF-R aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of BAFF-R aberrancy, for example,a BAFF-R agonist or BAFF-R antagonist agent can be used for treating thesubject. The appropriate agent can be determined based on screeningassays described herein.

Another aspect of the invention pertains to methods of modulating BAFF-Rexpression or activity for therapeutic purposes. The modulatory methodof the invention involves contacting a cell with an agent that modulatesone or more of the activities of BAFF-R protein activity associated withthe cell. An agent that modulates BAFF-R protein activity can be anagent as described herein, such as a nucleic acid or a protein, anaturally-occurring cognate ligand of a BAFF-R protein, a peptide, aBAFF-R peptidomimetic, or other small molecule. In one embodiment, theagent stimulates one or more BAFF-R protein activity. Examples of suchstimulatory agents include active BAFF-R protein and a nucleic acidmolecule encoding BAFF-R that has been introduced into the cell. Inanother embodiment, the agent inhibits one or more BAFF-R proteinactivity. Examples of such inhibitory agents include antisense BAFF-Rnucleic acid molecules and anti-BAFF-R antibodies. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of a BAFF-R protein or nucleic acidmolecule. In one embodiment, the method involves administering an agent(e.g., an agent identified by a screening assay described herein), orcombination of agents that modulates (e.g., upregulates ordownregulates) BAFF-R expression or activity. In another embodiment, themethod involves administering a BAFF-R protein or nucleic acid moleculeas therapy to compensate for reduced or aberrant BAFF-R expression oractivity.

In one embodiment, the invention provides methods of using BAFF-R.Included in such methods are methods of inhibiting B cell growth,dendritic cell-induced B cell growth and maturation or immunoglobulinproduction in an animal using BAFF-R polypeptide comprising at least aBAFF binding portion of BAFF-R. Other embodiments include methods ofstimulating B-cell growth, dendritic cell-induced B-cell growth andmaturation or immunoglobulin production in an animal using BAFF-Rpolypeptide (such as by transfecting cells which are deficient in BAFF-Rwith vectors to allow efficient expression of BAFF-R, or byadministering antibodies that bind BAFF-R and mimic BAFF).

In another embodiment, the invention provides methods of using BAFF-R inthe treatment of autoimmune diseases, hypertension, cardiovasculardisorders, renal disorders, B-cell lympho-proliferate disorders,immunosuppressive diseases, organ transplantation, and HIV. Alsoincluded are methods of using agents for treating, suppressing oraltering an immune response involving a signaling pathway between BAFF-Rand its ligand, and methods of inhibiting inflammation by administeringan antibody specific for a BAFF-R or an epitope thereof.

The methods of the present invention are preferably carried out byadministering a therapeutically effective amount of a BAFF-Rpolypeptide, a chimeric molecule comprising a BAFF-R polypeptide fusedto a heterologous amino acid sequence, or an anti-BAFF-R antibodyhomolog.

In one embodiment, the invention provides pharmaceutical compositionscomprising a BAFF-R polypeptide and a pharmaceutically acceptableexcipient.

In another embodiment, the invention provides chimeric moleculescomprising BAFF-R polypeptide fused to a heterologous polypeptide oramino acid sequence. An example of such a chimeric molecule comprises aBAFF-R fused to a Fc region of an immunoglobulin or an epitope tagsequence.

In another embodiment, the invention provides an antibody thatspecifically binds to a BAFF-R polypeptide. Optionally, the antibody isa monoclonal antibody.

In one embodiment of the invention is a method of treating a mammal fora condition associated with undesired cell proliferation byadministering to the mammal a therapeutically effective amount of acomposition comprising an BAFF-R antagonist, wherein the BAFF-Rantagonist comprises a polypeptide that antagonizes the interactionbetween BAFF-R and its cognate receptor or receptors, with apharmaceutically acceptable recipient.

In a preferred embodiment the cognate receptor of BAFF on the surface ofthe cell is BAFF-R.

The method can be used with any BAFF-R antagonist that has a polypeptidethat antagonizes the interaction between BAFF and its cognate receptoror receptors. Examples of BAFF-R antagonists include but are not limitedto soluble BAFF-R polypeptide, soluble chimeric BAFF-R molecules,including but not limited to BAFF-R-IgG-Fc and anti-BAFF-R antibodyhomologs.

The method of the invention can be used with any condition associatedwith undesired cell proliferation. In particular the methods of thepresent invention can be used to treat tumor cells which express BAFFand/or BAFF-R.

Examples of cancers whose cell proliferation is modulated by BAFF may bescreened by measuring in vitro the level of BAFF and/or BAFF-R messageexpressed in tumor tissue libraries. Tumor tissue libraries in whichBAFF and/or BAFF-R message is highly expressed would be candidates.Alternatively, one may screen for candidates searching the public andprivate databases (i.e., Incyte database) with, for example, the fulllength human BAFF cDNA sequence.

The BAFF-R antagonists of the subject invention which are used intreating conditions associated with undesired cell proliferation, inparticular tumor therapy, advantageously inhibit tumor cell growthgreater than 10%, 20%, 30% or 40% and most advantageously greater than50%. The BAFF-R antagonists are obtained through screening. For example,BAFF-R antagonists can be selected on the basis of growth inhibitingactivity (i.e., greater than 10%, 20%, 30%, 40% or 50%) against thehuman colon carcinoma HT29 or human lung carcinoma A549 which arederived from a colon and lung tumor respectively.

Another embodiment of the invention, provides methods of inhibitingB-cell and non-B cell growth, dendritic cell-induced B-cell growth andmaturation or immunoglobulin production in an animal using BAFF-Rpolypeptides such as those described above.

The method of inhibiting B-cell and non-B cell growth, dendriticcell-induced B-cell growth and maturation or immunoglobulin productionmay also include administration of an anti-BAFF-R antibody (polyclonalor monoclonal) that binds to BAFF-R and inhibits the binding of BAFF toBAFF-R. Administration of the antibody thereby inhibits B-cell and non-Bcell growth, dendritic cell-induced B-cell growth and maturation orimmunoglobulin production. The amount of antibody that may be suitablefor use may be extrapolated from the in vivo data provided herein.Various methods are known in the art to extrapolate dosages from animalexperiments, including for example, extrapolation based on body weightor surface area.

In some embodiments of the invention the BAFF-R:Fc polypeptides oranti-BAFF-R antibodies are administered in an amount of about 1 to 20mg/kg/dose. Doses may be given twice weekly, once weekly, one every twoweeks or once monthly, as needed. A physician will be able to determinethe proper dose by determining efficacy balanced against reducing anyuntoward effects of the therapy.

In another embodiment, the invention provides methods of using BAFF-R oranti-BAFF-R antibodies in the treatment of autoimmune diseases,hypertension, cardiovascular disorders, renal disorders, B-celllympho-proliferate disorders, immunosuppressive diseases, organtransplantation, inflammation, and HIV. Also included are methods ofusing agents for treating, suppressing or altering an immune responseinvolving a signaling pathway between BAFF-R and its ligand.

Methods of Inhibiting Aggregation of Expressed Protein, Including BAFF-Rand BAFF-R:Fc

The invention also provides a method for inhibiting or decreasingaggregation of expressed protein, particularly human BAFF-R orhuBAFF-R:Fc, which tends to aggregate during expression, frustratingpurification at high yields. In the method of the invention the aminoacid sequence of a protein that tends to aggregate when expressed in arecombinant system is compared to the amino acid sequence of a homologof the protein that exhibits less aggregation activity. The two homologswill have conserved domains and non-conserved amino acids there betweenand perhaps interspersed therein. In general, at least one of thenon-conserved amino acids amino acids of the aggregating protein may besubstituted for the amino acid in the homolog to alleviate aggregation.In some embodiments, nonpolar amino acids are substituted. Nonpolaramino acids include glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan, and cysteic. In someembodiments nonpolar amino acids substitute for other nonpolar aminoacids. Preferred nonpolar amino acids to inhibit or decrease aggregationare proline and alanine. In other embodiments, an uncharged polar aminoacid is substituted for a nonpolar amino acid. Uncharged polar aminoacids include asparagine, glutamine, serine, threonine and tyrosine.

In the method of the invention, substitutions are made that preferablyallow the protein to retain biological activity. In general,non-conserved amino acids are amenable to substitution withoutappreciably affecting biological activity.

In a specific example of the method of the invention, human BAFF-Rprotein may have amino acid substitutions introduced at positions V20,P21, A22 and L27 of SEQ ID NO:5 (or V41, P42, A43, and A48 of SEQ IDNO:10) and various combinations thereof, which greatly alleviatesaggregation of the protein. Similar strategies may be used for otherproteins that tend to aggregate when expressed in recombinant systems.While not wishing to be bound by any particular theory of operation, itis believed that the substitution of uncharged polar amino acids fornonpolar amino acids imparts solubility to the protein and discouragesaggregation of nonpolar regions between the proteins.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

EXAMPLES Example 1

This example describes the molecular cloning of BAFF-R, a novel receptorfor BAFF.

Materials and Methods

An oligo-dT primed cDNA library was made from BJAB cells, a human B cellline that binds human BAFF, and directionally cloned into the expressionvector CH269. CH269 is a derivative of pCEP4 (Invitrogen) that containsthe CMV promoter to drive expression of cloned DNA and also contains theoriP of EBV. This allows multicopy autonomous replication of theseplasmids in cells that are stably transformed with EBNA-1, such as293EBNA. The BJAB cDNA library was transfected into E. coli DH10B cellsand seeded in a 96 well format as pools of approximately 2500independent clones per well. DNA was prepared from these pools using theQiagen BioRobot 9600. The DNA pools were transfected using Lipofectamine(Life Technologies) into 293EBNA cells seeded into fibronectin coated 6well dishes. At 48 hours post-transfection the medium was removed andthe cells washed with plate assay wash buffer (20 mM HEPES, 0.5 mg/mlbovine serum albumin, 0.1% NaN₃). Cell monolayers were overlaid with 100mg/ml biotinylated human recombinant soluble myc-BAFF (myc-huBAFF) inbinding buffer (PBS, 2% fetal bovine serum, 0.1% NaN₃) and incubated atroom temperature for 1 hour. The myc-huBAFF (amino acids 136-285) usedin the assay was expressed in Pichia pastoris and purified by anionexchange chromatography followed by gel filtration.

The BAFF solution was removed and the cells were washed and fixed byincubation with 1.8% form aldehyde-0.2% glutaraldehyde in PBS for 5minutes. Cells were again washed and then incubated for 30 minutes withstreptavidin conjugated with alkaline phosphatase (SAV-AP) (JacksonImmunoResearch) at a 1:3000 dilution from stock in binding buffer. Cellswere washed and stained with fast red/napthol phosphate (Pierce). Cellsbinding the biotin-BAFF/SAV-AP complex were identified by the presenceof a red precipitate after inspection under low power microscopy.Secondary screening entailed plating out the DH10B glycerol stocks ofthe BAFF binding pools for single colonies, inoculating to culture inpools of 100, and repeating the BAFF binding assay as described above.Secondary screen positive pools were similarly broken down to individualclones and assayed for BAFF binding upon transfection to 293EBNA asdescribed above. The DNA sequence of the independent BAFF binding cloneswas determined.

Results

One of the BAFF binding clones was pJST576. It has in insert size of1201 base pairs (bp) not including the poly-A tail. The sequence of theinsert of pJST576 is shown in FIG. 1A (SEQ ID NO:1). BLAST analysis ofthis clone showed homology in the Genbank database to the chromosome 22BAC close HS250D10 (accession number Z99716). The entire pJST576sequence is found within this BAC. Homology was also found to the 3′ endof a human EST, AI250289 (IMAGE clone 2000271). The EST was generatedfrom a human follicular lymphoma library. The EST AI250289 was obtainedfrom Incyte and the sequence of the insert was determined (FIG. 1B) (SEQID NO:2). This sequence added 15 bp of 5′ sequence to the pJST576sequence, which is contiguous with the genomic sequence and 23 bp, whichwas not. The remainder of the EST sequence has perfect homology topJST576. An open reading frame could not be identified in these clones.

Example 2

In this example, we determine that the JST576 cDNA contains an intronand then establish an open reading frame.

Methods

The GENSCAN (Burge, C. & Karlin, S. J. (1997) Mol. Biol. 268:78-94) exonprediction program was run on the JST576 cDNA sequence. The results ofthis program predicted that an intron was present in the cDNA. In orderto determine if the prediction was correct, PCR analysis was performedon first strand cDNA from 2 cell lines expressing JST576. RNA waspurified from approximately 107 BJAB or IM-9 cells using the RNeasy kit(Qiagen) following the manufacturers suggested protocol. RNA wasquantitated and 5 μg was used for first strand cDNA reactions using theSuperscript preamplification kit (Life Technologies). Both oligo dT andrandom hexamers were used to generate the first strand product.Synthesis of the first strand was done following the recommendedprotocol. Three (I of each reaction, 10 ng of JST576 or no DNA was thenused as a template for PCR using oligonucleotides flanking the predictedintron. The oligonucleotides used in the reaction are the 5′ oligosBAF-225 [5′-GGCCGAGTGCTTCGACCTGCT-3′] (SEQ ID NO:33) or BAF-226[5′-GGTCCGCCACTGCGTGGCCTG-3′] (SEQ ID NO:34) and the 3′ oligo BAF-191[5′-CACCAAGACGGCCGGCCCTGA-3′] (SEQ ID NO:35). Each reaction contained1×Pfu buffer (Stratagene), 200 (M dNTPs, 10% DMSO, 150 ng of each oligo,and 1.25 units of Turbo Pfu polymerase (Stratagene). The reactions wererun for 35 cycles at 94° C. for 30 sec., 60° C. for 1 min. and 72° C.for 1.5 min. Ten μl of each reaction was run on a 1% agarose gel. Theremaining products from the BJAB and IM-9 BAF-225/191 reactions werepurified using the High Pure PCR product purification kit (RocheMolecular Biochemicals) and the bulk product was subjected to DNAsequencing. In addition, PCR products using the primers BAF-225 andBAF-191 were generated from resting B cell cDNA, subcloned andindividual clones were sequenced. Here 5 μl of resting B cell cDNA(Clontech) was used in a PCR reaction with the BAF-225 and BAF-191primers as detailed above. The PCR product was then purified using theHigh Pure PCR product purification kit and concentrated. In order tosubclone the PCR fragment, the ends of the fragment were phosphorylatedand made blunt using the Sure Clone ligation kit (Amersham PharmaciaBiotech) as recommended. The resulting product was cloned into the EcoRVsite of pBluescriptII (Stratagene) and transformed into E. coli.Individual colonies were grown up, the plasmid DNA miniprepped. Sixindependent isolates were sequenced.

Results

The mature nucleotide and amino acid sequence of JST576 predicted by theGENSCAN program is shown in FIG. 2A (SEQ ID NO:3). PCR products fromBJAB and IM-9 reactions spanning the predicted intron are shown in FIG.2B and confirm the existence of an intron in the JST576 cDNA clone. Thepredicted size of the PCR product from the JST576 cDNA is approximately788 bp for BAF-225/BAF-191 and 767 bp for BAF-226/BAF-191. The PCRproducts obtained from the JST576 template are approximately this size(lanes 10 and 11). The PCR products obtained using BAF-225BAF-191 oneither oligo dT primed BJAB or IM-9 first strand cDNA (lanes 2 and 6)are the same size and significantly shorter than the product from theJST576 cDNA. The predicted size of this fragment without the predictedintron is 484 bp. The size of the PCR products is consistent with thissize. The same results were obtained if BJAB or IM-9 RNA was primed withrandom hexamers (lanes 4 and 8). The reactions using BAF-226/BAF-191 didnot work on the first strand cDNA templates. Therefore, it appears thatthe intron predicted by the GENSCAN program does exist in the JST576cDNA. The sequence of the spliced product from BJAB and IM-9 RNA wasconfirmed by sequencing the bulk PCR product and is reflected in thesequence shown in FIG. 2C (SEQ ID NO:4). The sequence is identical tothe sequence shown in FIG. 2A (SEQ ID NO:3), except for the absence ofthe alanine codon (GCA) at nucleotide 149 (shown in small letters). Theresults of sequencing 6 independent clones from the RT-PCR reaction onresting B cell cDNA indicates that both splice acceptor sites areutilized. The preferred acceptor site appears to be the productresulting in one alanine residue (5/6 clones). However, the sequencepredicted by GENSCAN (SEQ ID NO:3), which contains two alanines, wasobserved in 1/6 clones. Therefore an open reading frame for human JST576has been established and a single amino acid splice variant has beendetermined. The open reading frame predicts a protein of 184 amino acidsshown in FIG. 2D (SEQ ID NO:5). The analine (A) residue in boldrepresents the splice variant. This protein is referred to as BAFF-R.The deduced amino acid sequence of BAFF-R includes a hydrophobic regionfrom residues 72-100 (Hopp-Woods algorithm) and a potentialtransmembrane segment from residues 84-102 as analyzed by the TMPredalgorithm. This region is followed by a highly charged stretch of aminoacids that may function as a stop transfer signal. BAFF-R lacks anN-terminal signal sequence and is a type III membrane protein similar tothe other BAFF binding proteins BCMA (Laabi et al. (1992) EMBO J.11:3897-3904) and TACI (von Bulow and Bram, (1997) Science 278:138-141).The N-terminus is predicted to be the extracellular domain of BAFF-R andcontains a 4 cysteine motif at residues 19-35 unlike any other member ofthe TNF receptor family. The C-terminus of BAFF-R is predicted to be theintracellular domain.

Example 3

Here we determine the DNA sequence upstream of the proposed initiatingmethionine for human BAFF-R including an in-frame stop codon.

Methods

A primer BAF-254 (5′GGGCGCCTACAATCTCAGCTA 3′) (SEQ ID NO:36) was made tothe genomic sequence present in the BAC HS250d10 (Genbank accessionnumber Z99716), upstream of the proposed ATG and used in a PCR reactionwith the oligo BAF-236 (5′ GGCGGACCAGCAGGTCGAAGCACTC 3′) (SEQ ID NO:37).The template in the reaction was first strand cDNA made from humanspleen RNA (Clontech) using the PCR preamplification kit as described bythe manufacturer (Life Technologies). The PCR reaction contained 3 μl ofthe first strand reaction, 1×Pfu buffer (Stratagene), 10% DMSO, 0.2 mMdNTPs, 150 ng each primer and 1.25 units Pfu Turbo polymerase(Stratagene). The PCR product was purified using the High Pure PCRProduct Purification kit following the manufacturer's directions (RocheMolecular Biochemicals). The ends of the PCR product were made blunt andphosphorylated using the Sure Clone ligation kit (Amersham PharmaciaBiotech), cloned into the EcoRV site of pBSK2 (Stratagene) andtransformed into DH5 cells. Colonies resulting from the ligation wereminiprepped using the Wizard system (Promega) and then sequenced usingan ABI machine.

Results

The sequence of the PCR product confirms that the mRNA containssequences directly upstream of the ATG that is contained in the genomicsequence. This sequence is underlined in the sequence shown in FIG. 3.The presence of an in-frame upstream stop codon and the absence ofanother methionine indicate that the methionine found in the JST576 cDNAis the correct initiating methionine.

Example 4

This example describes the cloning of the murine BAFF-R cDNA.

Methods

Approximately one million phage plaques were screened from the murineA20 cell line cDNA library purchased from Stratagene (La Jolla, Calif.)as detailed by the manufacturer. The JST576 human BAFF-R cDNA wasdigested with EcoNI and run on a 1% low melt gel. The 425 bp fragmentcontaining was cut out of the gel and weighed. Three times the volume ofwater was added and the gel fragment was boiled for 5 min. The fragmentwas labeled with 50 μCi ³²P-dCTP (Amersham) in a reaction containing 50mM Tris pH8, 5 mM MgCl₂, 10 μM β-mercaptoethanol, 200 mM HEPES pH 6.5,20 (M dNTPs (except dCTP), 0.27 units of pd(N)6 hexanucleotides(Amersham Pharmacia Biotech) and 1 unit of Klenow enzyme (USB) overnightat room temperature. About one million counts per mil of probe wasincubated with the filters in plaque screening buffer (50 mM Tris, 1%SDS, 1M NaCl, 0.1% Sodium Pyrophosphate, 0.2% PVP, 0.2% Ficoll, 0.2%BSA) overnight at 65° C. The filters were washed in 2×SSC and 0.1% SDSat 50° C. for 1.5 hrs (3×2 liters) and then exposed to x-ray film for 2days. Approximately 36 positive plaques were identified. Of these 6 wereplaque purified. The phagemids were released using the in vivo excisionprotocol detailed by Stratagene. The resulting colonies were grown upand the DNA was then miniprepped (Qiagen). The cDNA clones weresequenced.

Results

The murine BAFF-R consensus nucleotide sequence is presented as FIG. 4A(SEQ ID NO:8) and the amino acid sequence is presented in FIG. 4B (SEQID NO:9). Three of the clones contained a 10 amino acid deletion fromamino acid 119 to 129 in the intracellular domain of murine BAFF-R. Thealignment of the human and murine BAFF-R sequences illustrates that the4 cysteine residues in the extracellular domain are conserved, that theposition of the initiating methionine is similar and that the C-terminalregion of the proteins is highly conserved (FIG. 4C), with the last 24residues being identical. The sequences have approximately 56% identityoverall.

Example 5

In this example, the ability of human recombinant soluble BAFF to bindto cells co-transfected with pJST576 and a GFP reporter plasmid isdescribed.

Materials and Methods

The reporter plasmid encodes a membrane anchored GFP molecule and allowsidentification of transfected cells from non-transfected cells. 293EBNAcells were co-transfected with the reporter plasmid and pJST576 usingLipofectamine 2000 (Life Technologies). At 18-20 hr post-transfection,cells were detached from the plates with 5 mM EDTA in PBS and counted.The cells were washed twice with FACS buffer (PBS containing 10% fetalbovine serum, 0.1% NaN₃) and 2.5×10⁵ cells were incubated for 1 hour onice with biotinylated myc-huBAFF diluted into FACS buffer over aconcentration range of 8 ng/ml to 5 ug/ml. The cells were washed withFACS buffer and incubated for 30 minutes with streptavidin conjugatedwith phycoerythrin (SAV-PE) (Jackson ImmunoResearch) at a 1:100 dilutionfrom stock. The cells were again washed with FACS buffer and resuspendedin 1% paraformaldehyde in FACS buffer. The cells were analyzed by FACSfor GFP and PE fluorescence and the data was formatted in a fourquadrant dot plot. The dots in the two rightward quadrants representcells expressing the transfection reporter GFP. The dots in the twoupper quadrants represent cells having bound biotinylated myc-hBAFF withthis binding revealed by SAV-PE. The cells in the upper right quadrantare transfected cells that bind biotinylated myc-huBAFF.

Results

Unstained cells and cells stained only with SAV-PE show approximately50% are GFP positive and have been co-transfected with the reporterplasmid (FIG. 5). When cells co-transfected with the GFP reporter andpJST576 are stained with 1 ug/ml biotinylated myc-huBAFF nearly all thecells in the lower right quadrant shift up, indicating BAFF binding. Asimilar result is seen if a plasmid expressing huTACI is co-transfectedin place of pJST576. TACI is known to bind BAFF. The cells were stainedwith five fold dilutions of biotinylated myc-huBAFF from 5 ug/ml to 8ng/ml and as the concentration of biotinylated myc-huBAFF decreased theintensity of the shift decreased.

Example 6

In this example, the ability of human recombinant soluble BAFF or murinerecombinant soluble BAFF to bind to cells co-transfected with pJST576and a GFP reporter plasmid is described.

Materials and Methods

Co-transfections to 293EBNA were as described in Example 5. At 18-20 hrpost-transfection, cells were detached, counted, and stained for FACSanalysis similar to Example 5 with the following modifications. Thecells were incubated for 1 hour on ice with 5 ug/ml of either murine orhuman recombinant soluble flag-BAFF, followed after washing byincubation for 30 minutes with 5 ug/ml of the anti-flag monoclonalantibody M2 (Sigma Aldrich), and then revealed by incubating the washedcells for 30 minutes with PE conjugated donkey anti-mouse IgG (JacksonImmunoResearch) at a 1:100 dilution from stock. The cells were againwashed, fixed with paraformaldehyde, and analyzed by FACS for GFP and PEpositive cells.

Results

Approximately 50% of the cells are GFP positive and have therefore beenco-transfected with the reporter plasmid (FIG. 6). When cellsco-transfected with the GFP reporter and pJST576 are stained with 5ug/ml of either human or murine recombinant soluble flag-BAFF, nearlyall the cells in the lower right quadrant shift up. This indicates thatboth murine and human BAFF bind to cells transfected pJST576.

Example 7

In this example, the inability of murine recombinant soluble APRIL tobind to cells co-transfected with pJST576 and a GFP reporter plasmid isdescribed.

Materials and Methods

Co-transfections to 293EBNA were as described in Example 5. At 18-20 hrpost-transfection, cells were detached, counted, and stained for FACSanalysis similar to Example 5 with the following modifications. Thecells were incubated for 1 hour on ice with 1 ug/ml of murinerecombinant soluble myc-APRIL, followed after washing by incubation for30 minutes with 5 ug/ml of anti-murine APRIL monoclonal antibody,followed by a 30 minute incubation of the washed cells with 5 ug/mlbiotinylated anti-rat IgG2b (Pharmingen), and finally revealed byincubating the washed cells for 30 minutes with SAV-PE. The cells wereagain washed, fixed with paraformaldehyde, and analyzed by FACS for GFPand PE positive cells.

Results

Approximately 50% of the cells are GFP positive and have therefore beenco-transfected with the reporter plasmid (FIG. 7). When cellsco-transfected with the GFP reporter and pJST576 are stained with 1ug/ml of murine myc-APRIL, none of the cells in the lower right quadrantshift up. This is in contrast to cells co-transfected with a plasmidexpressing human TACI instead of pJST576. In these transfected cells,nearly all were positive for murine myc-APRIL binding. It has beenpreviously shown that both BAFF and APRIL bind to both TACI and BCMA.Therefore the fact that APRIL does not bind to BAFF-R as expressed onpJST576 transfected cells indicates a specificity of BAFF-R for BAFF.

Example 8

This example describes the ability of BAFF-R as expressed from pJST576to be co-immunoprecipitated by recombinant soluble human flag-BAFF.

Materials and Methods

293EBNA cells were transfected by Lipofectamine 2000 with pJST576, avector only control, or a plasmid expressing huTACI as a positivecontrol for BAFF binding. After 20 hours incubation the transfectionmedium was aspirated, the cells washed with PBS, and the media replacedwith ³⁵S labeling media (9 parts DMEM lacking methionine and cysteine to1 part complete DMEM, supplemented with 10% dialyzed fetal bovine serum,4 mM glutamine, and 100 μCi/ml ³⁵S methionine and cysteine (Translabel,ICN Radiochemicals). Cells were incubated in this medium for six hoursafter which the media was removed. Cells were washed with PBS and thensolubilized with 250 μl Extraction Buffer (1% Brij 98, 150 mM NaCl, 50mM Tris pH7.5). Co-immunoprecipitations were performed by incubating 75μl of the ³⁵S labelled cell extracts with 5 μg recombinant soluble humanflag-BAFF in 1 ml DMEM-10% fetal bovine serum-0.1% NaN₃ overnight at 4°C. The anti-flag monoclonal antibody M2, 10 μg, and protein A-Sepharosewere added and incubations continued for 2 hours. The Sepharose beadswere collected by centrifugation, washed with FACS buffer, andresuspended in SDS loading buffer with beta-mercaptoethanol as areducing agent. Samples were boiled 5 minutes, centrifuged briefly topellet the Sepharose beads, and an aliquot run on SDS-PAGE. The gel wasincubated with Enlightning (New England Nuclear), dried down, andexposed to film at −80° C.

Results

This co-immunoprecipitation binds flag-BAFF to the protein A Sepharosebeads through the anti-flag antibody, M2. It will also bring down anyproteins in the cell extract that bind to flag-BAFF, and theseradiolabelled proteins will be detected by autoradiography. As 293EBNAcells do not bind BAFF, the empty vector control shows the backgroundinherent in the procedure (FIG. 8). When extracts from cells transfectedfor TACI are co-immunoprecipitated with flag-BAFF, a band with anapparent molecular weight of approximately 34 kDa is observed. This isthe approximate predicted molecular weight for full length human TACI(31.2 kDa), a protein known to bind BAFF. When extracts from cellstransfected with pJST576 are co-immunoprecipitated with flag-BAFF, aband with an apparent molecular weight of approximately 12 kDa isobserved. The predicted molecular weight for BAFF-R expressed frompJST576 is 18.9 kDa. The disparity between predicted and observedmolecular weights could be due to anomalous electrophoretic mobility dueto the charge or conformation of BAFF-R. Another possibility is that 12kDa is a proteolytic fragment of BAFF-R.

Example 9

This example describes the generation of soluble forms of BAFF-R.Oligonucleotide primers complementary to pJST576 can be designed to PCRamplify the BAFF-R extracellular domain in the absence of transmembraneand intracellular domains. Typically, one includes most of the stalk, oramino acid region between the ligand binding domain and thetransmembrane domain. One could vary amount of stalk region included tooptimize the potency of the resultant soluble receptor. This amplifiedfragment would be engineered with suitable restriction sites to allowcloning to various heterlogous leader sequences on the 5′ end of thefragment and to various Ig fusion chimera fusion vectors at the 3′ end.Alternatively, one can insert a stop signal at the 3′ end of the BAFF-Rextracellular domain to and make a soluble form of the receptor or useanother C-terminal fusion partner without resorting to the use of aninstead of using the Ig fusion chimera approach. Also, one could createan N-terminal fusion protein consisting of a fusion partner containing asignal sequence followed by the N-terminal extracellular domain ofBAFF-R. The resultant vectors can be expressed in most systems used inbiotechnology including yeast, insect cells, bacteria, and mammaliancells and examples exist for all types of expression Various human Fcdomains can be attached to optimize or eliminate FcR and complementinteractions as desired. Alternatively, mutated forms of these Fcdomains can be used to selectively remove FcR or complement interactionsor the attachment of N-linked sugars to the Fc domain which has certainadvantages. An example of a BAFF-R:Fc fusion molecule is shown in FIG.9. This molecule contains the type I leader sequence from a murine Ig-kgene linked by an Aat2 restriction site to the BAFF-R extracellulardomain (amino acid residues 2-71 as shown in FIG. 2D) which is in turnlinked by a SalI restriction site to the Fc domain of human IgG1.

Example 10

In this example we show the expression profile of BAFF-R in humantissues and cell lines using Northern blot analysis.

Materials and Methods

Various B and non-B cell lines were grown under the appropriateconditions. RNA was prepared from approximately 10⁷ cells using theRNeasy kit (Qiagen). The RNAs were quantified and 20 μg of each samplewas run on a 1.2% formaldehyde gel as described by Sambrook et al.MOLECULAR CLONING: A LABORATORY MANUAL, 1989. The gel was blotted to anylon membrane (BMB) and then ultraviolet (UV) cross-linked. Severalhuman Northern blots (12 lane multi-tissue, human II and immune systemII) were purchased from Clontech. The filters was were prehybridized at65° C. in ExpressHyb (Clontech) buffer for 30 min. and then hybridizedwith a randomly primed ³²P labeled EcoNI fragment from the 3′ end ofJST576 for about 3 hrs. The filters were washed at room temperature in2×SSC/0.05% SDS for 45 min. and then at 50° C. in 0.1×SSC/0.1% SDS for45 min. The filter was exposed to X-ray film for 4 days using 2intensifying screens. In addition, several human Northern blots (12 lanemulti-tissue, human II and Immune system II) were purchased fromClontech, hybridized to the JST576 probe and treated as above.

Results

The mRNA for BAFF-R appears to predominately expressed in the immunesystem organs at this level of detection. The highest level is in thespleen and lymph nodes, but mRNA was also apparent in PBLs, thymus,small intestine and colon (FIGS. 10 A, B and C). The approximate size ofthe message is 4.5 kb; there appears to be two mRNA populations in thesamples where the gene is not highly expressed. Two mRNAs may exist inthe spleen and lymph nodes as well. This may indicate that BAFF-R hasalternative polyA addition sites or that the RNA undergoes alternativesplicing. When a number of cell lines were examined for the presence ofBAFF-R mRNA, the same 4.5 kb mRNA is detected. Only B cell lines expressBAFF-R mRNA (FIG. 11). No mRNA is detected in the U266, RPMI8226 andDaudi cell lines or in the non-B cell lines examined.

Example 11

In this example we show that JST576 expression is restricted to the celllines that bind BAFF.

Materials and Methods

Cell lines were purchased from ATCC and grown under the indicatedconditions. Various B and non-B cell lines were grown under theappropriate conditions. RNA was prepared from approximately 10⁷ cellsusing the RNeasy kit (Qiagen). The RNAs were quantitated and 20 μg ofeach sample was run on a 1.2% formaldehyde gel as described by Sambrooket al. MOLECULAR CLONING: A LABORATORY MANUAL, 1989. The gel was blottedto a nylon membrane (BMB) and then U cross-linked. The filter washybridized with a JST576 labeled fragment and then washed as in Example10. The cells were checked for their ability to bind BAFF using FACSanalysis. Approximately 2.5-5×10⁵ cells were collected, and washed.FLAG-tagged BAFF, diluted in PBS+5% FCS and 0.05% sodium azide (FACSbuffer), was incubated with the cells over the concentration range(8-0.125 μg/ml) for 30 min. on ice. The cells were washed with FACSbuffer and incubated, for 30 min. on ice, with the anti-FLAG monoclonalantibody M2 (Sigma) at 5 μg/ml. Again the cells were washed with FACSbuffer and then incubated with a 1:5000 dilution of goat anti-mouse IgGPE conjugated antibody (Jackson Immuno Research) for 30 min. on ice. Thecells were washed as above and then analyzed on a FACSCalibur flowsorter (Becton-Dickinson) using CellQuest software.

Results

The results of the BAFF binding experiments are shown in Table 1. Thecell lines that bind BAFF are Ramos, Namalwa, IM-9, NC-37, Raji, BJABand SKW6.4. The level of binding is indicated by the number of + signs.The cell lines that do not bind BAFF are U266, RPMI 8226, Daudi, U937,Jurkat, HT29, A549, SW480 and ME260. The ability of the cell lines tobind BAFF is correlated to the presence of BAFF-R mRNA shown in FIG. 11.TABLE 1 Cell Line Type BAFF Binding BJABIM-9 Burkitt lymphoma +++Lymphoblast IgG +++ NC-37 Lymphoblast EBV+ ++ Ramos Burkitt lymphomaEBV− ++ Raji Burkitt lymphoma ++ SKW6.4 Lymphoblast IgM ++ NamalwaBurkitt lymphoma + Daudi Burkitt lymphoma EBV+ − U266 Plasmacytoma −RPMI 8226 Plasmacytoma − U937 Monocyte − Jurkat T Cell leukemia − HT29Colorectal − adenocarcinoma A549 Lung carcinoma − SW480 Colorectal −adenocarcinoma ME260 Melanoma −

Example 12

This example describes the ability of a huBAFF-R:huIgG1 fusion proteinthat is expressed and secreted into the conditioned media by transientlytransfected 293EBNA cells to co-immunoprecipitate recombinant solublebiotinylated myc-huBAFF.

Materials and Methods

293EBNA cells were transfected by Lipofectamine-2000 (LifeTechnologies)with either pJST618 which expresses huBAFF-R (aa2-71):Fc, a plasmidexpressing huBCMA:huIgG1 as a positive control for BAFF binding, or aplasmid expressing huFN14:huIgG1 as a negative control for BAFF binding.After 24 hours incubation the conditioned media was harvested.

SDS-PAGE was run by mixing an equal volume of 2×SDS running buffer, withor without reducing agent, with the conditioned media and boiling for 5minutes. The samples were then run on a 4-20% SDS polyacrylamide gel.Known quantities of purified hBCMA:Fc were run in adjacent lanes toestimate amount of hIgG1 fusion protein in the conditioned media.Samples were transferred to membranes (Immunobillon P, Millipore) bywestern blot in 0.01M CAPS pH11-10% MeOH buffer. Membranes were blockedwith 5% non-fat dry milk (NFDM) in TBST, probed with 1:3000 dilution ofgoat anti-human IgG-HRP (Jackson ImmunoResearch) for 1 hour, washed inTBST and exposed to film. Co-immunoprecipitations were performed byincubating 200 μl of the conditioned media with 200 ng recombinantsoluble human flag-BAFF in 1 ml DMEM-10% fetal bovine serum-0.1% NaN3overnight at 4° C. Protein A-Sepharose was added and incubationscontinued for 2 hours. The Sepharose beads were collected bycentrifugation, washed with FACS TBST buffer, and resuspended in SDSloading buffer with beta mercaptoethanol as a reducing agent. Sampleswere boiled 5 minutes, centrifuged briefly to pellet the Sepharosebeads, and an aliquot run on SDS-PAGE. FLAG-huBAFF, 50 ng, was run as apositive control. Samples were transferred to PVDF membranes(Immunobillon P, Millipore) by western blot in 0.01M CAPS pH 11/10% MeOHbuffer. Membranes were blocked with 5% NFDM-TBST, probed with 1 μg/mlanti-FLAG M2-HRP for 1 hour, washed in TBST and exposed to film.

Results

Co-immunoprecipitation brings down the various receptor:Fc fusionsthrough the fusion partner interacting with protein A Sepharose. It willalso bring down any proteins interacting with the R:IgG1 fusions, suchas the flag-BAFF. Conditioned media from cells expressing hBCMA:Fc areable to co-immunoprecipitate flag-BAFF, as expected, as a band thatco-migrates with flag-BAFF is observed on the western blot (FIG. 12).Conditioned media from cells expressing hFN14:Fc does notco-immunoprecipitate flag-BAFF. The conditioned media from cellsexpressing BAFF-R:Fc are able to co-immunoprecipitate flag-BAFF. A bandthat co-migrates with flag-BAFF is observed on the western blot and isof similar intensity to that co-immunoprecipitated by huBCMA:huIgG1.

Example 13

This example illustrates the ability of a BAFF-R:Fc fusion protein, inthis case huBAFF-R (aa2-71):huIgG1, to block the binding of huBAFF toBJAB cells.

Materials and Methods

The huBAFF-R (2-71)-huIgG1 fusion discussed in example 9 was generatedand called pJST618. This construct was transiently transfected into293EBNA cells and the conditioned media was harvested. The fusionprotein was purified by acid elution from proteinA Sepharose followed byand gel filtration chromatography. Biotinylated myc-huBAFF, 200 ng/ml,was preincubated with either 50 ul FACS buffer or with five fold serialdilutions, ranging from 5 μg/ml to 200 ng/ml, of purified huBAFF-R:Fcfor 30 minutes on ice. BJAB cells (2.5×10⁵ cells) were then incubatedwith these solutions on ice for one hour. Cells were washed with FACSbuffer and stained with SAV-PE. The cells were analyzed by FACS for PEfluorescence and the data was formatted as overlayed histograms.Alternatively, 200 ng/ml biotinylated-BAFF was pre-incubated withtwo-fold serial dilutions of either hBAFF-R:Fc, hTACI:Fc, or hLTBR:Fc.Cells were stained for biotinylated BAFF binding as above.

Results

FIG. 13A shows the overlay of the histograms plotted for huBAFF bindingto BJAB in the presence of various concentrations of huBAFF-R:Fc. Theblack line labelled “A” represents background binding of SAV-PE and thered line marked “E” represents cells stained with biotinylatedmyc-huBAFF without pre-incubation with BAFF-R:Fc. Pre-incubation ofbiotinylated myc-huBAFF with 5 μg/ml of huBAFF-R:Fc results in a shiftin the histogram nearly to background levels (curve B). Pre-incubationwith either 1 μg/ml (curve C) or 200 ng/ml (curve D) huBAFF-R-huIgG1resulted in an approximate four-fold decrease in biotinylated myc-huBAFFbinding.

FIG. 13B shows that both BAFF-R:Fc and TACI:Fc are able to block BAFFbinding to BJAB cells. Pre-incubation with LTBR:Fc has no BAFF blockingeffect.

Example 14

This example describes the ability of a BAFF-R:IgG1 fusion protein toblock BAFF-induced B cell proliferation.

Material and Methods

For the in vitro proliferation assay, mouse B cells were isolated fromspleens of C57B16 mice (8 weeks old) using a B cell recovery column(column (Cellect™ Mouse B Cell Recovery Column: Cedarlane LaboratoriesLimited, Ontario, Canada). Purified B cells were analyzed by FACSand >90% were found positive for B220 staining. B cells were incubatedin 96-well plates (10⁵ cells/well in 50 ml RPMI supplemented with 10%FBS) for 72 hours in the presence or absence of 2 mg/ml of goatanti-human m chain antibody (Sigma Chemical Co.); control hIgG (10mg/ml) huBAFF-R:Fc (10 mg/ml). The samples were done plated intriplicate and with the indicated concentrations of myc-hBAFF. Cellswere pulsed for an additional 18 hours with [³H]thymidine (1 μCi/well)and harvested. [³H]Thymidine incorporation was monitored by liquidscintillation counting. Human BAFF-R:Fc fusion protein, produced as inexample 13, was used in this assay, as discussed in example 9, wasgenerated from the supernatant of pJST618 transfected 293EBNA cells. Thesupernatant was harvested, loaded onto a Protein A column, eluted withacid, neutralized and then subjected to gel filtration chromatography inorder to obtain aggregate-free huBAFF-R:Fc protein. The BAFF used in theassay was expressed in Pichia pastoris and purified by anion exchangechromatography followed by gel filtration.

Results

FIG. 14 shows that BAFF can costimulate B cell growth in the presence ofanti-m antibodies (squares) and hIgG (triangles). BAFF alone (diamonds)is not able to induce B cell proliferation. Incubation with 10 mg/ml ofhuBAFF-R:Fc (stars) results in a complete inhibition of BAFF-induced Bcell proliferation.

Materials and Methods

Mice

Six-week old female BALB/c mice were obtained from The JacksonLaboratory (Bar Harbor, Me.) and maintained under barrier conditions inthe Biogen Animal Facility.

Reagents and Treatment Regimen

Receptor fusion proteins contain the human IgG1 Fc region. Mice(5/group) received 200 μg of fission proteins (mouse BAFF-R:Fc or humanBAFF-R:Fc) 2×/week for 4 weeks, ip (intraperitoneally). Control micereceived polyclonal human IgG (Panglobulin™) (HIgG), 200 μg 2×/week for4 weeks. Three days after the last dose, blood was collected via theorbital sinus, then mice were euthanized and spleens, lymph nodes, andbone marrow were collected for analysis.

Flow Cytometric Analysis

At the time of sacrifice spleen weights were recorded. Single cellsuspensions were prepared from spleen and blood after lysing red bloodcells in a hypotonic solution. Single cell suspensions were alsoprepared from inguinal lymph nodes and bone marrow. Flow cytometry wasperformed using mAbs directed against B220, IgM, IgD and CD21. Splenic Bcell subpopulations were defined as follicular (B220+, IgM^(low),CD21^(low)), marginal zone (B220+, IgM^(hi) CD21^(hi)) and newly formed(B220+, IgM^(hi) CD21−). Briefly, ˜1.5×10⁶ cells were incubated with 10μg/ml of Fc Block (Pharmingen) for 10 min on ice to block Fc receptors,followed by addition of fluorescently tagged mAbs and incubated on icefor 30 min. Cells were washed 1× and resuspended in 0.5%paraformaldehyde. Cell fluorescence data were acquired on a FACSCaliburflow cytometer (Becton Dickinson, San Jose, Calif.) and analyzed usingCellQuest software (Becton Dickinson).

Results

After a 4-week treatment course with mouse or human BAFF-R:Fc there wasa marked reduction in the weight of spleens from mice treated with mouseand human BAFF-R:Fc (FIG. 15), as compared to control Human IgG-treatedmice. The apparent decline in splenic cellularity was found to resultfrom a reduction in the number of splenic B cells. The mean number oftotal B220+ splenic B cells in mouse and human BAFF-R:Fc-treated mice,1.8×10⁶ and 2.6×10⁶ cells, respectively, was significantly reduced whencompared to the number of B cells in control HIgG-treated animals, whichhad a mean of 19.8×10⁶ cells (FIG. 16). Examination of differentsubpopulations of splenic B cells, follicular, marginal zone and newlyformed, indicated that the number of B cells in each subset was reducedin the BAFF-R::Fc-treated mice (Table 2), although follicular andmarginal zone B cells had the greatest reduction. TABLE 2 BAFF-R::FcTreatment Results in a Reduction in Splenic B Cell SubpopulationsSplenic B cell subpopulations (10⁶ cells ± SD) Follicular Marginal ZoneNewly formed Human IgG 14.5 ± 2.4  1.1 ± 0.3 1.5 ± 0.2 mBAFF-R:Fc 0.7 ±0.1 0.06 ± 0.02 0.4 ± 0.1 hBAFF-R:Fc 1.4 ± 0.5 0.05 ± 0.02 0.5 ± 0.2Mice received 200 μg of HIgG, mBAFF-R:Fc or hBAFF-R:Fc on days 1, 4, 8,11, 15, 18, 22 and 25. Mice were euthanized on day 28 and spleens wereharvested for analysis of B cell subsets.

Examination of the percent of B220+ B cells contained in inguinal lymphnodes (LN) showed that the mean B cell populations were markedly reducedin mouse and human BAFF-R::Fc-treated mice, 12.3%±1.4 and 18.6%±1.3,respectively, when compared to control HIgG-treated mice which had amean of 30.8%±4.1 B cells (FIG. 17). Similar results were obtained whenperipheral blood B cells were examined. 42.5%±2.9 of the lymphocytesfrom human IgG-treated mice were B cells, whereas only 21.2%±6.1 and8.3%±4.5 of lymphocytes were B cells from mouse and humanBAFF-R::Fc-treated mice, respectively (FIG. 18).

Although newly formed (immature) B cell and mature B cell populationswere reduced in BAFF-R::Fc-treated mice, B cell precursors in the bonemarrow remained unaffected (data not shown).

Discussion

These results suggest that in vivo blockade of BAFF with a solubleBAFF-R receptor fusion protein leads to the inhibition of B cellsurvival and/or maturation.

These results also suggest the potential use of a BAFF-R fusion proteinas a therapeutic drug with clinical applications in B cell-mediateddiseases. Diseases would include those that are autoimmune in naturesuch as systemic lupus erythematosus, myasthenia gravis, autoimmunehemolytic anemia, idiopathic thrombocytopenia purpura, anti-phospholipidsyndrome, Chagas' disease, Grave's disease, Wegener's granulomatosis,poly-arteritis nodosa and rapidly progressive glomerulonephritis. Thistherapeutic agent would also have application in plasma cell disorderssuch as multiple myeloma, Waldenstrom's macroglobulinemia, heavy-chaindisease, primary or immunocyte-associated amyloidosis, and monoclonalgammopathy of undetermined significance (MGUS). Oncology targets wouldinclude B cell carcinomas, leukemias, and lymphomas.

Example 16

In this example, the characterization of an initial panel of mousemonoclonal antibodies raised against the extracellular domain of BAFF-Ris described. All antibodies recognize the extracellular domain ofBAFF-R, and a subset of these antibodies have antagonist properties inthat they prevent subsequent binding of BAFF to BAFF-R.

Materials and Methods:

RBF mice were immunized and boosted with huBAFF-R:Fc. Splenocytes fromthe immune mouse were fused with mouse myeloma strain FL653, aderivative of strain P3-X63-Ag8.653 to generate hybridomas by standardtechnologies.

Conditioned media from hybridoma clones secreting antibodies against theextracellular domain of huBAFFR were assayed by FACS. FACS bindingassays were performed 293EBNA cells co-transfected with plasmidsexpressing full length huBAFF-R or muBAFF-R and GFP as in Example 5.Hybridoma conditioned media was diluted 1:10 in FACS buffer andincubated with the transfected cells 30 min on ice. Cells were washedwith FACS buffer and binding was revealed by incubation with a 1:100dilution of anti-mouse IgG (H+L) (Jackson ImmunoResearch) for 30 min onice. The cells were again washed with FACS buffer and resuspended in 1%paraformaldehyde in FACS buffer. The cells were analyzed by FACS for GFPand PE fluorescence and the data was formatted in a four quadrant dotplot as described in Example 5. BAFF blocking assays were performed byincubating 10 ug/ml proteinA purified anti-BAFF-R mAb or controlantibody (MOP C21) with BJAB cells for 30 min on ice. After washing,cells were incubated with 250 ng/ml biotinylatedhuBAFF for 30 min onice. Cells were again washed and BAFF binding was revealed by incubationwith SAV-PE. The cells were analyzed by FACS for PE fluorescence anddata was plotted as overlayed histograms.

Results:

The supernatants from ten clones were observed to bind huBAFF-Rtransfected cells. The dot plots of the FACS data of four of the tenanti-BAFF-R supernatants are shown in FIG. 19A. Transfection efficiencywas approximately 50%, with nearly all transfected cells shifting to theupper right quadrant after staining with supernatants. None of these tensupernatants bound to 293EBNA cells transfected with muBAFFR (data notshown). Conditioned media from the clones that were positive for bindingto BAFF-R were tested for their ability to block the interaction of BAFFwith the BAFF-R expressed on the surface of BJAB cells. BJAB cellsexpress BAFFR on their surface, and express no detectable amounts ofBCMA or TACI (Thompson et al. (2001) Science August 16). Two of the tenhybridomas, clones 2 and 9, produced mAbs that were able to block theinteraction of BAFF-R with BAFF. (Clone 2 was deposited with the ATCC onSep. 6, 2001 as “anti-BAFF-R clone #2.1” (IgG1-kappa isotype) and hasbeen assigned ATCC No. PTA-3689; Clone 9 was deposited with the ATCC onSep. 6, 2001 as “anti-BAFF-R clone #9.1” (IgG1-kappa isotype) and hasbeen assigned ATCC No. PTA-3688). The overlays of the histograms in FIG.19B show that preincubation of 10 μg/ml of either mAb clone 2 (curve(b)) or 9 (curve (c)) shifts the BAFF binding curve greater thanten-fold to the left, nearly to the signal of the no BAFF control (curve(a)). The rightmost histogram (curve (d)) indicates the shift whencontrol mAb MOP C21, anti-BAFF-R non-blocking mAbs, or no protein wereincubated with the cells prior to BAFF binding.

Example 17

This example describes the construction, sequence and proteincharacterization of amino acid substitutions in hBAFF-R(2-71)-Fc thatresult in increased solubility of the recombinantly expressed molecule.

Materials and Methods:

Double stranded oligonucleotide cassettes with cohesive ends were usedto introduce substitutions at targeted residues by ligation into thesame sites in the hBAFF-R(2-71):IgG1 gene.

Expression plasmids were transfected into 293EBNA cells usingLipofectamine 2000 as in Example 5. Aggregation was determined byrunning non-reducing SDS-PAGE of 20 hr post-transfection conditionedmedia, followed by western transfer, and detection with HRP conjugatedanti-human IgG (1:100, Jackson ImmunoResearch) and ECL detection as inExample 12.

Immunoprecipitation experiments were performed utilizing 100 μl of 20 hrpost-transfection conditioned media in 1 ml of DMEM/10% FBS/0.2% NaA3with 200 ng flag-huBAFF. Samples were rocked for 30 min at 4° C., 30 ulprotein A-Sepharose was added per tube and rocking continued for another30 minutes. Sepharose beads were spun down and washed three times with 1ml cold PBS. Beads were resuspended in 2×SDS reducing buffer and loadedonto 4-20% acrylamide gels. After western transfer as previouslydescribed, the ability to immunoprecipitate flag-BAFF was revealed byincubation of the filters with 1 μg/ml HRP conjugated anti-flag M2(Sigma) followed by ECL detection.

Results:

While the human BAFF-R:Fc is highly aggregated, the murine BAFF-R:Fc isonly slightly (<10%) aggregated. Deletion analysis has shown that theentire C-terminal BAFF-R moiety can be deleted from A71 to V36 (last Cysof Cysteine Rich Domain (CRD) is C35) with no decrease in aggregateformation. This would implicate the N-terminal and CRD regions of hBAFFRas being required for aggregate formation.

Initially, several murine-human BAFF-R:Fc chimeras were generated inwhich various amounts of N-terminal human BAFF-R sequence were replacedwith the homologous murine sequence and analyzed for the effect onprotein aggregation. The amino acid sequence for these and subsequentsubstitutions into hBAFF-R:Fc are shown in FIG. 20. This figure showsthe BAFF-R moiety of both the “wild type” human (FIG. 9) and murineBAFF-R:Fc, with the numbering corresponding to the amino acid residuesfrom the full length human (FIG. 2 d) (SEQ ID NO:5) or murine BAFF-R(FIG. 4 b) (SEQ ID NO:9). FIG. 20 also shows the hBAFFR-R:Fc clones withsubstitutions, with the substituted residues indicated in bolded, red,underlined type. The chimeras containing less than the first 21 murineresidues (Q21) before switching over to human appear to aggregatesimilar to wild type hBAFF-R:Fc; however, those that contain at leastthe first 39 murine residues aggregate in a markedly reduced manner,similar to mBAFF-R. Of the additional nine residues different betweenthese two chimeric BAFF-R:Fc constructs, four of them differ betweenmouse and human. This would implicate at least one of the human residuesbetween C19 and L27, a region internal to the CRD, as being required foraggregation.

Constructs replacing the human residues with those corresponding tomurine at only these 4 sites or a subset thereof were made by standardtechniques. When only the 4 residues V20N P21Q A22T L27P weresubstituted into the human BAFFR moiety, this modified BAFF-R:Fc was notaggregated. hBAFF-R(V20N P21Q A22T L27P):Fc were still able to interactwith BAFF as analyzed by immunoprecipitation. The V20N L27P substitutionalso reduced aggregation of hBAFF-R:Fc from approximately 90% to about10%. Intermediate levels of aggregation were observed with P21Q L27P(40%), L27P (60%), V20N L27A (60%) and V20N L27S (60%). None of thefollowing substitutions diminished protein aggregation: V20N P21Q A22T;V20N A22T; V20N P21Q; V20N; and P21Q.

Example 18

This example describes p21-Arc is a protein associated with BAFF-R. Themethod used to determine such an interaction was immunoprecipitation.

Methods

A construct containing the cDNA encodes the intracellular domain ofBAFF-R (BAFF-R-i.c.d.) with a myc tagged fused at the N-terminus wasmade and subcloned into CH269 plasmid at NheI (5′) and XhoI (3′) sites.The 293E cells were tranasfected with this construct and were lysed 72hours after with lysis buffer containing in 150 mM NaCl, 50 mM Tris-HCl,pH7.5, 1 mM Na3VO4, 50 mM NaF and 1% Brij 97. The cell lysates werecleared with a table top centrifuge at 10,000 g for 5 minutes and wereimmunoprecipitated with an anti-myc monoclonal antibody, 9E10. Theimmunoprecipitates were separated by a 10-20% SDS-PAGE under reducingconditions and were trans-blotted onto a PVDF membrane. The blottedproteins were visualized with 0.2% Ponceau S solution and the areascorresponding to proteins specifically associated with BAFF-R wereexcised and subjected to N-terminal amino acid sequence analysis. Anambiguous search in the non-redundant protein database using the PATTERNSEARCH algorithm was performed for the obtained N-terminal sequencedata.

Results

One of the proteins specifically associated with the myc-tagged BAFFRcytoplasmic domain has an apparent molecular weight of 21 kDa. Thisprotein was unambiguously identified as the p21-Arc (Actin relatedprotein complex). P21-Arc is a component of a seven subunits proteincalled Arp2/3 complex which was shown to be involved in the actinpolymerization Welch et al. (1997) J. Cell Biol. 138:357). Recently, anactin-binding protein, filamin, was reported to be associated with thetumor necrosis factor receptor-associated factor 2 (TRAF2) (Leonardi etal. (2000) J. Biol. Chem. 275:271). Thus, the identification of p21-Arcin the co-immunoprecipitates of BAFFR cytoplasmic domain suggestsp21-Arc is either directly associated the BAFFR or indirectly associatedwith BAFFR via its association with TRAF2 and/or other TRAF proteinwhich, in turn, associates with the BAFFR.

From the foregoing detailed description of the specific embodiments ofthe invention, it should be apparent that unique have been described.Although particular embodiments have been disclosed herein in detail,this has been done by way of example for purposes of illustration only,and is not intended to be limiting with respect to the scope of theappended claims which follow. In particular, it is contemplated by theinventor that various substitutions, alterations, and modifications maybe made to the invention without departing from the spirit and scope ofthe invention as defined by the claims.

1-55. (canceled)
 56. A method of detecting a BAFF-R polypeptide, saidmethod comprising: (a) providing a biological sample; and (b) contactingthe sample with an isolated antibody, or an antigen binding portionthereof, that specifically binds to an amino acid sequence selected fromthe group consisting of SEQ ID NO:5, SEQ ID NO:10, and SEQ ID NO:9. 57.The method of claim 56, wherein the antibody is chimeric.
 58. The methodof claim 56, wherein the antibody is humanized.
 59. The method of claim56, wherein the antibody is monoclonal.
 60. The method of claim 56,wherein the antibody is a single-chain antibody.
 61. The method of claim56, wherein the antibody is a Fab fragment.
 62. The method of claim 56,wherein the antibody is as produced by hybridoma clone #2.1, depositedunder ATCC No. PTA-3689.
 63. The method of claim 56, wherein theantibody is as produced by hybridoma clone #9.1, deposited under ATCCNo. PTA-3688.
 64. A method of measuring BAFF-R mRNA levels, said methodcomprising: (a) providing a biological sample from a subject; and (b)determining the level of BAFF-R mRNA in the sample using a nucleic acidmolecule capable of hybridizing under stringent conditions to: (i) anucleic acid sequence that encodes an amino acid sequence selected fromthe group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID SEQ ID NO:9and SEQ ID NO:10; or (ii) a complement of (ii).
 65. The method of claim64, wherein the nucleic acid molecule is a oligonucleotide probeconsisting of at least 15, 30, 50, 100, 250, or 500 consecutivenucleotides from: (a) a complement of a nucleic acid sequence thatencodes SEQ ID NO:9 or SEQ ID NO:10; or (b) a nucleic acid sequence thatencodes SEQ ID NO:9 or SEQ ID NO:10.
 66. The method of claim 65, whereinthe oligonucleotide probe is labeled.
 67. The method of claim 64,wherein the method comprises the step of using polymerase chain reaction(PCR).
 68. The method of claim 56 or claim 64, wherein the biologicalsample is a tissue, fluid or cell from a subject.
 69. The method ofclaim 68, wherein the BAFF-R mRNA encodes the amino acid sequence of SEQID NO:13.
 70. The method of claim 69, wherein the BAFF-R mRNA encodesthe amino acid sequence of SEQ ID NO:10.
 71. The method of claim 56 orclaim 64, wherein the BAFF-R mRNA encodes the amino acid sequence of SEQID NO:14.
 72. The method of claim 71, wherein the BAFF-R mRNA encodesthe amino acid sequence of SEQ ID NO:9.
 73. The method of claim 56 orclaim 64, wherein the biological sample is obtained from a mammal. 74.The method of claim 73, wherein the mammal is human.
 75. The method ofclaim 73, wherein the mammal has or is at risk of having a disease orcondition selected from the group comprising a B-cell-mediatedcondition, an autoimmune condition, a tumorigenic condition, a plasmacell disorder, a B-cell carcinoma, leukemia, lymphoma, hypertension, acardiovascular disorder, a renal disorder, an immunosuppressivedisorder, organ transplantation, and Burkitt's lymphoma.
 76. The methodof claim 75, wherein the mammal has or is at risk for B cell carcinoma,leukemia, or lymphoma.
 77. The method of claim 75, wherein theautoimmune condition is systemic lupus erythematosus.
 78. The method ofclaim 75, wherein the autoimmune condition is rheumatoid arthritis. 79.The method of claim 73, wherein the mammal has or is at risk of having adisease or condition selected from multiple myeloma, Waldenstrom'smacroglobulinemia, heavy-chain disease, primary or immunocyte associatedamyloidosis, or monoclonal gammopathy of undetermined significance(MGUS).
 80. A method of diagnosing a disease or condition in a subjectcomprising the method of claim 56, thereby diagnosing the disease orcondition.
 81. A method of diagnosing a disease or condition in asubject comprising the method of claim 57, thereby diagnosing thedisease or condition.
 82. A method of diagnosing a B-cell mediatedcondition in a subject comprising: (a) providing a biological samplefrom the subject; (b) measuring the amount of BAFF-R polypeptide in thesubject sample; and (c) comparing the amount of BAFF-R polypeptide inthe subject sample to the amount of BAFF-R polypeptide in a controlsample, wherein a difference in the amount of BAFF-R polypeptide in thesubject sample as compared to the amount of BAFF-R polypeptide in thecontrol sample indicates that the subject has a B-cell mediatedcondition.
 83. A method of diagnosing a B-cell-mediated condition in asubject comprising: (a) measuring the amount of BAFF-R nucleic acid in asample obtained from the subject; and (b) comparing the amount of BAFF-Rnucleic acid in the subject sample to the amount of BAFF-R nucleic acidin a control sample, wherein a difference in the amount of BAFF-Rnucleic acid in the subject sample as compared to the amount of BAFF-Rnucleic acid in the control sample indicates that the subject has aB-cell-mediated condition.
 84. A method for identifying cells or tissuesthat misexpress BAFF-R comprising: (a) contacting an oligonucleotideprobe comprising at least 25 consecutive nucleotides from any one of thefollowing: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQID NO:6 with a biological sample of a subject; and (b) measuring a levelof a BAFF-R-encoding nucleic acid molecule in the biological sample bydetecting BAFF-R mRNA levels; or alternatively, (c) determining whethera BAFF-R nucleic acid molecule in the biological sample has been mutatedor deleted.
 85. A method for determining whether a genomic BAFF-Rsequence has been mutated or deleted comprising: (a) providing abiological sample of a subject; and (b) determining whether a BAFF-Rnucleic acid molecule in the biological sample has been mutated ordeleted using an nucleic acid molecule capable of hybridizing with humanor mouse genomic sequence of BAFF-R.